Regulation of thrombopoietin receptor expression and function

Of the many cells in the body, the hematopoietic cells are among those with the highest rate of self-renewal and turnover. The production and destruction of these cells are tightly controlled by a number of hematopoietic growth factors, in particular by members of the family of helical cytokines. Studying the thrombopoietin receptor, I focused on two aspects of cytokine receptor signaling: attenuation of signaling by receptor isoforms and the biological function of cytokine receptor target genes. Cytokine receptor signaling has profound effects on cell survival, proliferation and differentiation. It is therefore not surprising that components of the signaling cascade are tightly regulated at the level of expression. An important mechanism for controlling gene expression is alternative splicing. Alternate isoforms have been identified for many cytokine receptors and a regulatory function and/or altered expression in disease have been described for some of these isoforms The cytokine thrombopoietin (TPO) and its cognate receptor c-mpl are the primary regulators of platelet production and also play an important role in hematopoietic stem cell biology. Several isoforms of unknown function exist for both mouse and human mpl and it is possible that they play an important role in modulating mpl signaling. In my thesis work, I have analyzed the function of a truncated receptor isoform (mpl-tr) which is the only alternate mpl isoform conserved between mouse and humans. Although mpl-tr lacks a transmembrane domain, classifying it as a ‘secreted’ or ‘soluble receptor’, it is retained intracellularly. My results provide evidence that mpl-tr acts as a dominant-negative variant of mpl for both proliferation and survival. I also demonstrate that mpl-tr mediates protein degradation of the full-length receptor by a cathepsin-like cysteine protease activity. Due to a shift of the reading frame at a splice acceptor site, the C-terminus of mpl-tr consists of a peptide of unique sequence, 30 amino acids in length. I show that this peptide sequence is essential for the inhibition of TPO-dependent proliferation and for mpl protein degradation mediated by mpl-tr. Together, these data suggest a new paradigm for the regulation of cytokine receptor expression and function through a proteolytic process directed by a truncated isoform of the same receptor. To test for the in vivo function of alternative mpl isoforms, a c-mpl cDNA was expressed as a transgene in mpl knockout mice. These mice express mpl fulllength as the only mpl isoform and develop severe thrombocytosis with platelet numbers, elevated about five times higher than normal. The reintroduction of the endogenous mpl allele restores normal platelet counts and I attribute this to the in vivo effect of dominant-negative mpl isoforms. A mpl knock-in allele, which does not express mpl-tr but still expresses the second known alternate variant of murine mpl, mpl-II, normalizes platelet numbers, similar to the endogenous mpl allele. This result demonstrates that the absence of mpl-tr is not sufficient to cause thrombocytosis. I propose that mpl-II is an additional dominant-negative mpl isoform and attenuates the expansion of the megakarocytic lineage in vivo. In summary, these results impressively demonstrate the importance of alternate cytokine receptor isoforms in vivo and emphasize the need to study the function of the many uncharacterized cytokine receptor isoforms. In a second project, I studied the role of mpl signaling in regulating the expression of a gene with a potential role in cell differentiation and proliferation. The diversification of cell types is controlled through the use of both lineage-restricted and more widely expressed transcriptional regulators and the combinatorial actions of these regulators specify gene expression. The differentiation of megakaryocyte precursors is dependent on the proper function of the GATA-1 transcription factor. Mice lacking GATA-1 selectively in megakaryocytes have dramatically fewer platelets but more megakaryocytes, altered platelet size and shape and prolonged bleeding times. Further, GATA- 1-null megakaryocytes hyperproliferate in vitro, suggesting that GATA-1 is both a differentiation factor and negative regulator of megakaryocyte cell proliferation. However, GATA-1 regulated genes which are responsible for this growth inhibition are presently unknown. In this thesis work, I describe a novel gene, GASIP (GATA-1 regulated SIAH Interacting Protein), which is dramatically downregulated in mpl-transfected hematopoietic cell lines, identifying mpl as a negative regulator of GASIP expression. The presence of juxtaposed GATA and Ets-binding cis-elements in the GASIP promoter are typical for a megakaryocytic gene. I found that GASIP expression in platelets is indeed robust and correlates with mRNA levels of GATA-1, but not GATA-2 or –3, identifying GATA-1 as a positive regulator of GASIP expression. The finding that mpl and GATA have opposite effects on both proliferation and on GASIP expression, make GASIP a candidate GATA-1 target gene involved in growth inhibition. To investigate the potential role of GASIP in growth regulation, I screened for potential protein binding partners. Interestingly, I identified the p53-inducible tumor suppressor seven in absentia homologe (SIAH) as a GASIP interacting protein. I speculate that GASIP may contribute to the anti-proliferative effect mediated by SIAH

Molecular Pathology of Lewy Body Diseases

Lewy body diseases are characterized by the presence of Lewy bodies, alpha-synuclein(AS)-positive inclusions in the brain. Since their main component is conformationally modified AS, aggregation of the latter is thought to be a key pathogenic event in these diseases. The analysis of inclusion body constituents gives additional information about pathways also involved in the pathology of synucleinopathies. Widespread mitochondrial dysfunction is very closely related to disease development. The impairment of protein degradation pathways, including both the ubiquitin-proteasome system and the autophagy-lysosome pathway also play an important role during the development of Lewy body diseases. Finally, differential expression changes of isoforms corresponding to genes primarily involved in Lewy body formation point to alternative splicing as another important mechanism in the development of Parkinson’s disease, as well as dementia with Lewy bodies. The present paper attempts to give an overview of recent molecular findings related to the pathogenesis of Lewy body diseases

Functional Studies of Deubiquitinating Enzymes

The attachment of ubiquitin to substrate proteins is a key process in regulating cellular events such as cell cycle progression, signal transduction, differentiation, apoptosis, and the clearance of misfolded or aberrant proteins. Like other post-translational modifications, ubiquitination is also reversible. Deconjugation is performed by a family of cysteine- or metallo-proteases collectively known as deubiquitinating enzymes (DUBs). Approximately 100 putative DUBs have been identified in the human genome but only a minority of them has been functionally characterized. The aim of this thesis has been to study the function of selected DUBs in disease-relevant cellular pathways. Screen of the canonical Wnt-signaling pathway with an RNA interference (RNAi) library targeting the human DUBs identified the ubiquitin-specific protease (USP)-4 as a negative regulator. USP4 interacts with two known components in the pathway: the Nemo like kinase (Nlk) and the T-cell factor 4 (TCF4). NLK promotes nuclear accumulation of USP4 where a subpopulation of TCF4 is a substrate of USP4-dependent deubiquitination. Using a yeast-2 hybrid strategy to search for relevant interactions, we identified the proteasome as a binding partner of USP4. USP4 interacts with the S9 subunit of the 19S regulatory particle (RP) through an N-terminal ubiquitin-like (UBL) domain that resembles, but is functionally distinct from, the UBLs of hHR23a/b and Ubiquilin-1. S9 is as an essential proteasome subunit that may regulate the structural integrity of the 26S complex. Thus, USP4 may play a role in the dynamics of ubiquitination at the proteasome. A bioinformatics strategy was used to search for membrane-associated DUBs. We found that a putative transmembrane domain targets USP19 to the endoplasmic reticulum (ER). USP19 is a target of the unfolded protein response and rescues ERAD substrate from proteasomal degradation. Moreover, USP19 interacts with the E3 ligases seven in absentia homolog (SIAH) 1 and SIAH2 that mediate USP19 ubiquitination and degradation by the proteasome. Bioinformatics and biochemical analysis revealed the presence in USP19 of a SIAH-interacting motif that is found in a subset of SIAH targets and may function as a degradation signal. A non-enzymatic role of USP19 in the regulation of the reposed to hypoxia was suggested by the finding that wild-type and catalytic mutant USP19 interact with the hypoxia-inducible factor-1α (HIF-1α). In the absence of USP19, cells fail to mount a proper response to hypoxia

Vascular fibrosis in aging and hypertension: molecular mechanisms and clinical implications

Aging is the primary risk factor underlying hypertension and incident cardiovascular disease. With aging, the vasculature undergoes structural and functional changes characterized by endothelial dysfunction, wall thickening, reduced distensibility, and arterial stiffening. Vascular stiffness results from fibrosis and extracellular matrix (ECM) remodelling, processes that are associated with aging and are amplified by hypertension. Some recently characterized molecular mechanisms underlying these processes include increased expression and activation of matrix metalloproteinases, activation of transforming growth factor-β1/SMAD signalling, upregulation of galectin-3, and activation of proinflammatory and profibrotic signalling pathways. These events can be induced by vasoactive agents, such as angiotensin II, endothelin-1, and aldosterone, which are increased in the vasculature during aging and hypertension. Complex interplay between the “aging process” and prohypertensive factors results in accelerated vascular remodelling and fibrosis and increased arterial stiffness, which is typically observed in hypertension. Because the vascular phenotype in a young hypertensive individual resembles that of an elderly otherwise healthy individual, the notion of “early” or “premature” vascular aging is now often used to describe hypertension-associated vascular disease. We review the vascular phenotype in aging and hypertension, focusing on arterial stiffness and vascular remodelling. We also highlight the clinical implications of these processes and discuss some novel molecular mechanisms of fibrosis and ECM reorganization

Role of nuclear bodies in apoptosis signalling

AbstractPromyelocytic leukemia nuclear bodies (PML NBs) are dynamic macromolecular multiprotein complexes that recruit and release a plethora of proteins. A considerable number of PML NB components play vital roles in apoptosis, senescence regulation and tumour suppression. The molecular basis by which PML NBs control these cellular responses is still just beginning to be understood. In addition to PML itself, numerous further tumour suppressors including transcriptional regulator p53, acetyl transferase CBP (CREB binding protein) and protein kinase HIPK2 (homeodomain interacting protein kinase 2) are recruited to PML NBs in response to genotoxic stress or oncogenic transformation and drive the senescence and apoptosis response by regulating p53 activity. Moreover, in response to death-receptor activation, PML NBs may act as nuclear depots that release apoptotic factors, such as the FLASH (FLICE-associated huge) protein, to amplify the death signal. PML NBs are also associated with other nuclear domains including Cajal bodies and nucleoli and share apoptotic regulators with these domains, implying crosstalk between NBs in apoptosis regulation. In conclusion, PML NBs appear to regulate cell death decisions through different, pathway-specific molecular mechanisms

Comprehensively Surveying Structure and Function of RING Domains from Drosophila melanogaster

Using a complete set of RING domains from Drosophila melanogaster, all the solved RING domains and cocrystal structures of RING-containing ubiquitin-ligases (RING-E3) and ubiquitin-conjugating enzyme (E2) pairs, we analyzed RING domains structures from their primary to quarternary structures. The results showed that: i) putative orthologs of RING domains between Drosophila melanogaster and the human largely occur (118/139, 84.9%); ii) of the 118 orthologous pairs from Drosophila melanogaster and the human, 117 pairs (117/118, 99.2%) were found to retain entirely uniform domain architectures, only Iap2/Diap2 experienced evolutionary expansion of domain architecture; iii) 4 evolutionary structurally conserved regions (SCRs) are responsible for homologous folding of RING domains at the superfamily level; iv) besides the conserved Cys/His chelating zinc ions, 6 equivalent residues (4 hydrophobic and 2 polar residues) in the SCRs possess good-consensus and conservation- these 4 SCRs function in the structural positioning of 6 equivalent residues as determinants for RING-E3 catalysis; v) members of these RING proteins located nucleus, multiple subcellular compartments, membrane protein and mitochondrion are respectively 42 (42/139, 30.2%), 71 (71/139, 51.1%), 22 (22/139, 15.8%) and 4 (4/139, 2.9%); vi) CG15104 (Topors) and CG1134 (Mul1) in C3HC4, and CG3929 (Deltex) in C3H2C3 seem to display broader E2s binding profiles than other RING-E3s; vii) analyzing intermolecular interfaces of E2/RING-E3 complexes indicate that residues directly interacting with E2s are all from the SCRs in RING domains. Of the 6 residues, 2 hydrophobic ones contribute to constructing the conserved hydrophobic core, while the 2 hydrophobic and 2 polar residues directly participate in E2/RING-E3 interactions. Based on sequence and structural data, SCRs, conserved equivalent residues and features of intermolecular interfaces were extracted, highlighting the presence of a nucleus for RING domain fold and formation of catalytic core in which related residues and regions exhibit preferential evolutionary conservation

TBLR1 regulates the expression of nuclear hormone receptor co-repressors

BACKGROUND: Transcription is regulated by a complex interaction of activators and repressors. The effectors of repression are large multimeric complexes which contain both the repressor proteins that bind to transcription factors and a number of co-repressors that actually mediate transcriptional silencing either by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes. RESULTS: TBLR1 [GenBank: NM024665] is a co-repressor of nuclear hormone transcription factors. A single highly conserved gene encodes a small family of protein molecules. Different isoforms are produced by differential exon utilization. Although the ORF of the predominant form contains only 1545 bp, the human gene occupies ~200 kb of genomic DNA on chromosome 3q and contains 16 exons. The genomic sequence overlaps with the putative DC42 [GenBank: NM030921] locus. The murine homologue is structurally similar and is also located on Chromosome 3. TBLR1 is closely related (79% homology at the mRNA level) to TBL1X and TBL1Y, which are located on Chromosomes X and Y. The expression of TBLR1 overlaps but is distinct from that of TBL1. An alternatively spliced form of TBLR1 has been demonstrated in human material and it too has an unique pattern of expression. TBLR1 and the homologous genes interact with proteins that regulate the nuclear hormone receptor family of transcription factors. In resting cells TBLR1 is primarily cytoplasmic but after perturbation the protein translocates to the nucleus. TBLR1 co-precipitates with SMRT, a co-repressor of nuclear hormone receptors, and co-precipitates in complexes immunoprecipitated by antiserum to HDAC3. Cells engineered to over express either TBLR1 or N- and C-terminal deletion variants, have elevated levels of endogenous N-CoR. Co-transfection of TBLR1 and SMRT results in increased expression of SMRT. This co-repressor undergoes ubiquitin-mediated degradation and we suggest that the stabilization of the co-repressors by TBLR1 occurs because of a novel mechanism that protects them from degradation. Transient over expression of TBLR1 produces growth arrest. CONCLUSION: TBLR1 is a multifunctional co-repressor of transcription. The structure of this family of molecules is highly conserved and closely related co-repressors have been found in all eukaryotic organisms. Regulation of co-repressor expression and the consequent alterations in transcriptional silencing play an important role in the regulation of differentiation

Components of the Ubiquitin Proteasome System are Required for the Nonapoptotic Death of the C. Elegans Linker Cell

Cell death is a major cell fate that promotes tissue sculpting and morphogenesis during animal development. Many developmental cell-culling events cannot be accounted for solely by caspase-dependent apoptosis, yet, alternate pathways are poorly understood. Direct evidence that caspase-independent non-apoptotic cell death pathways operate during animal development is provided by studies of the C. elegans linker cell. Genetic studies of linker cell death have led to the identification of genes that promote this process, including pqn-41, which encodes a glutamine-rich protein, as well as tir-1/TIRdomain and sek-1/MAPKK, which may function in the same pathway as pqn-41. The let- 7 microRNA and its indirect target, the Zn-finger transcription factor LIN-29, also promote linker cell death, and may act early in the process. Our work suggests that components of the ubiquitin proteasome system (UPS) act to promote linker cell death. We show that LET-70, an E2 ubiquitin-conjugating enzyme, is required cell-autonomously for linker cell death. LET-70 levels, as well as those of ubiquitin and some proteasome components, increase just before linker cell death initiation. This rise is dependent on an MLL-type histone methyltransferase complex and a MAPK cascade, whose activities are required for linker cell death. The E3 ligase components SIAH-1, RBX-1, and CUL-3 are also required for linker cell death and appear to act in the same pathway as let-70. We also identify the PLZF transcription factor EOR-1, and its accessory protein, EOR-2 as major regulators of linker cell death. Our studies suggest that EOR-1/2, as well as all known regulators of the linker cell death pathway may act upstream of UPS components to promote cell death. Our studies reveal that activation of the ubiquitin proteasome system is an important event promoting linker cell death. Given the morphological similarities between linker cell death and non-apoptotic developmental and pathological cell death in vertebrates, we raise the possibility that the proteasome may be a key mediator of vertebrate cell death

Multiple, but Concerted Cellular Activities of the Human Protein Hap46/BAG-1M and Isoforms

The closely related human and murine proteins Hap46/BAG-1M and BAG-1, respectively, were discovered more than a decade ago by molecular cloning techniques. These and the larger isoform Hap50/BAG-1L, as well as shorter isoforms, have the ability to interact with a seemingly unlimited array of proteins of completely unrelated structures. This problem was partially resolved when it was realized that molecular chaperones of the hsp70 heat shock protein family are major primary association partners, binding being mediated by the carboxy terminal BAG-domain and the ATP-binding domain of hsp70 chaperones. The latter, in turn, can associate with an almost unlimited variety of proteins through their substrate-binding domains, so that ternary complexes may result. The protein folding activity of hsp70 chaperones is affected by interactions with Hap46/BAG-1M or isoforms. However, there also exist several proteins which bind to Hap46/BAG-1M and isoforms independent of hsp70 mediation. Moreover, Hap46/BAG-1M and Hap50/BAG-1L, but not the shorter isoforms, can bind to DNA in a sequence-independent manner by making use of positively charged regions close to their amino terminal ends. This is the molecular basis for their effects on transcription which are of major physiological relevance, as discussed here in terms of a model. The related proteins Hap50/BAG-1L and Hap46/BAG-1M may thus serve as molecular links between such diverse bioactivities as regulation of gene expression and protein quality control. These activities are coordinated and synergize in helping cells to cope with conditions of external stress. Moreover, they recently became markers for the aggressiveness of several cancer types

Engineering aldo-keto reductase 1B10 to mimic the distinct 1B15 topology and specificity towards inhibitors and substrates, including retinoids and steroids

The aldo-keto reductase (AKR)superfamily comprises NAD(P)H-dependent enzymes that catalyze the reduction of a variety of carbonyl compounds. AKRs are classified in families and subfamilies. Humans exhibit three members of the AKR1B subfamily: AKR1B1 (aldose reductase, participates in diabetes complications), AKR1B10 (overexpressed in several cancer types), and the recently described AKR1B15. AKR1B10 and AKR1B15 share 92% sequence identity, as well as the capability of being active towards retinaldehyde. However, AKR1B10 and AKR1B15 exhibit strong differences in substrate specificity and inhibitor selectivity. Remarkably, their substrate-binding sites are the most divergent parts between them. Out of 27 residue substitutions, six are changes to Phe residues in AKR1B15. To investigate the participation of these structural changes, especially the Phe substitutions, in the functional features of each enzyme, we prepared two AKR1B10 mutants. The AKR1B10 m mutant carries a segment of six AKR1B15 residues (299-304, including three Phe residues)in the respective AKR1B10 region. An additional substitution (Val48Phe)was incorporated in the second mutant, AKR1B10mF48. This resulted in structures with smaller and more hydrophobic binding pockets, more similar to that of AKR1B15. In general, the AKR1B10 mutants mirrored well the specific functional features of AKR1B15, i.e., the different preferences towards the retinaldehyde isomers, the much higher activity with steroids and ketones, and the unique behavior with inhibitors. It can be concluded that the Phe residues of loop C (299-304)contouring the substrate-binding site, in addition to Phe at position 48, strongly contribute to a narrower and more hydrophobic site in AKR1B15, which would account for its functional uniqueness. In addition, we have investigated the AKR1B10 and AKR1B15 activity toward steroids. While AKR1B10 only exhibits residual activity, AKR1B15 is an efficient 17-ketosteroid reductase. Finally, the functional role of AKR1B15 in steroid and retinaldehyde metabolism is discussed