Riccardo Sgarra

Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia

The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound βE/β0-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The βE-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated βE-globin with partial instability. When this is compounded with a non-functional β0 allele, a profound decrease in β-globin synthesis results, and approximately half of βE/β0-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral β-globin gene transfer, an adult patient with severe βE/β0-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl−1, of which one-third contains vector-encoded β-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

Nuclear phosphoproteins HMGA and their relationship with chromatin structure and cancer

The structural characteristics of the three nuclear phosphoproteins of the high mobility group A family are outlined and related to their participation in chromatin structure alteration in many biological processes such as gene expression, neoplastic transformation, differentiation, and apoptosis. The elevated expression of these proteins in tumor cells and their post‐translational modifications, such as phosphorylation, acetylation and methylation, are discussed and suggested as suitable targets for cancer chemotherapy.

Transcriptional Activation of the Cyclin A Gene by the Architectural Transcription Factor HMGA2

ABSTRACT The HMGA2 protein belongs to the HMGA family of architectural transcription factors, which play an important role in chromatin organization. HMGA proteins are overexpressed in several experimental and human tumors and have been implicated in the process of neoplastic transformation. Hmga2 knockout results in the pygmy phenotype in mice and in a decreased growth rate of embryonic fibroblasts, thus indicating a role for HMGA2 in cell proliferation. Here we show that HMGA2 associates with the E1A-regulated transcriptional repressor p120E4F, interfering with p120E4F binding to the cyclin A promoter. Ectopic expression of HMGA2 results in the activation of the cyclin A promoter and induction of the endogenous cyclin A gene. In addition, chromatin immunoprecipitation experiments show that HMGA2 associates with the cyclin A promoter only when the gene is transcriptionally activated. These data identify the cyclin A gene as a cellular target for HMGA2 and, for the first time, suggest a mechanism for HMGA2-dependent cell cycle regulation.

The AT-hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-methylated by Protein Arginine Methyltransferase 6

The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg57 and Arg59, two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization.

HMGA molecular network: From transcriptional regulation to chromatin remodeling.

Nuclear functions rely on the activity of a plethora of factors which mostly work in highly coordinated molecular networks. The HMGA proteins are chromatin architectural factors which constitute critical hubs in these networks. HMGA are referred to as oncofetal proteins since they are highly expressed and play essential functions both during embryonic development and neoplastic transformation. A particular feature of HMGA is their intrinsically disordered status, which confers on them an unusual plasticity in contacting molecular partners. Indeed these proteins are able to bind to DNA at the level of AT-rich DNA stretches and to interact with several nuclear factors. In the post-genomic era, and with the advent of proteomic tools for the identification of protein-protein interactions, the number of HMGA molecular partners has increased rapidly. This has led to the extension of our knowledge of the functional involvement of HMGA from the transcriptional regulation field to RNA processing, DNA repair, and chromatin remodeling and dynamics. This review focuses mainly on the protein-protein interaction network of HMGA and its functional outcome. HMGA molecular partners have been functionally classified and all the information collected in a freely available database (http://www.bbcm.units.it/ approximately manfiol/INDEX.HTM).

HMGA1 is a novel downstream nuclear target of the insulin receptor signaling pathway

High-mobility group AT-hook 1 (HMGA1) protein is an important nuclear factor that activates gene transcription by binding to AT-rich sequences in the promoter region of DNA. We previously demonstrated that HMGA1 is a key regulator of the insulin receptor (INSR) gene and individuals with defects in HMGA1 have decreased INSR expression and increased susceptibility to type 2 diabetes mellitus. In addition, there is evidence that intracellular regulatory molecules that are employed by the INSR signaling system are involved in post-translational modifications of HMGA1, including protein phosphorylation. It is known that phosphorylation of HMGA1 reduces DNA-binding affinity and transcriptional activation. In the present study, we investigated whether activation of the INSR by insulin affected HMGA1 protein phosphorylation and its regulation of gene transcription. Collectively, our findings indicate that HMGA1 is a novel downstream target of the INSR signaling pathway, thus representing a new critical nuclear mediator of insulin action and function.

Conformational role for the C-terminal tail of the intrinsically disordered high mobility group A (HMGA) chromatin factors.

The architectural factors HMGA are highly connected hubs in the chromatin network and affect key cellular functions. HMGA have a causal involvement in cancer development; in fact, truncated or chimeric HMGA forms, resulting from chromosomal rearrangements, lack the constitutively phosphorylated acidic C-terminal tail and display increased oncogenic potential, suggesting a functional role for this domain. HMGA belong to the intrinsically disordered protein category, and this prevents the use of classical approaches to obtain structural data. Therefore, we combined limited proteolysis, ion mobility separation-mass spectrometry (IMS-MS), and electrospray ionization-mass spectrometry (ESI-MS) to obtain structural information regarding full length and C-terminal truncated HMGA forms. Limited proteolysis indicates that HMGA acidic tail shields the inner portions of the protein. IMS-MS and ESI-MS show that HMGA proteins can assume a compact form and that the degree of compactness is dependent upon the presence of the acidic tail and its constitutive phosphorylations. Moreover, we demonstrate that C-terminal truncated forms and wild type proteins are post-translationally modified in a different manner. Therefore, we propose that the acidic tail and its phosphorylation could affect HMGA post-translational modification status and likely their activity. Finally, the mass spectrometry-based approach adopted here proves to be a valuable new tool to obtain structural data regarding intrinsically disordered proteins.

Macroscopic Differences in HMGA Oncoproteins Post-Translational Modifications: C-Terminal Phosphorylation of HMGA2 Affects Its DNA Binding Properties

HMGA is a family of nuclear proteins involved in a huge number of functions at the chromatin level. It consists of three members, HMGA1a, HMGA1b, and HMGA2, having high sequence homology and sharing the same structural organization (three highly conserved DNA-binding domains, an acidic C-terminal tail, and a protein-protein interaction domain). They are considered important nodes in the chromatin context, establishing a complex network of interactions with both promoter/enhancer sequences and nuclear factors. They are involved in a plethora of biological processes and their activities are finely tuned by several different post-translational modifications. We have performed an LC/MS screening on several different cell lines to investigate HMGA proteins expression and their post-translational modifications in order to detect distinctive modification patterns for each. Our analyses evidenced relevant macroscopic differences in the phosphorylation and methylation patterns of these proteins. These differences occur both within the HMGA family members and in the different cell types. Focusing on HMGA2, we have mapped its in vivo phosphorylation sites demonstrating that, similarly to the HMGA1 proteins, it is highly phosphorylated on the acidic C-terminal tail and that these modifications affect its DNA binding properties.

Interaction proteomics of the HMGA chromatin architectural factors

The high mobility group A (HMGA) chromatin architectural transcription factors are a group of proteins involved in development and neoplastic transformation. They take part in an articulated interaction network, both with DNA and other nuclear proteins, organizing multimolecular complexes at chromatin level. Here, we report the development of a novel in vitro strategy for the identification of HMGA molecular partners based on the combination of an RP‐HPLC prefractionation procedure, 2‐DE gels, blot‐overlay and MS. To demonstrate that our approach could be a reliable screening method we confirmed a representative number of interactions in vitro by GST pull‐down and far‐Western and in vivo by co‐affinity purification. This approach allowed us to enlarge the HMGA molecular network confirming their involvement also in non‐transcriptional‐related processes such as RNA processing and DNA repair.

Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex

The three‐dimensional structures of the native cytochrome c2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 Å and in the reduced state at 1.95 Å resolution. These are the first high‐resolution crystal structures in both oxidation states of a cytochrome c2 with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six‐residue insertion Gly82‐Ala87, which precedes the heme binding Met93, forms an isolated 310‐helix secondary structural element not previously observed in other c‐type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c2 with other c‐type cytochromes has revealed that the presence of such a residue, with torsion angles ϕ and ξ of approximately −140 and −130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 Å resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X‐ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high‐pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c‐type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H‐bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c2 of the rare six‐residue insertion in the helix 310 conformation that increases Met loop flexibility.