Staying tuned for post-COVID-19 syndrome: looking for new research to sniff out.

Post-COVID-19 syndrome was defined as a persistent and protracted illness, which follows acute COVID-19 infection. This condition continues for more than 12 weeks and cannot be attributed to other clinical situations. Researchers and clinicians are allied in unraveling the molecular pathogenetic mechanisms and the clinical development of this unexpected SARS-CoV-2 infectious evolution. Anosmia, dysgeusia, fatigue, dyspnea, and 'brain fog' are common symptoms observed in the Post-COVID-19 syndrome, depicting a multiorgan involvement associated with injuries involving mainly cardiovascular, pulmonary, musculoskeletal, and neuropsychiatric systems. This commentary analyzes the state of the art of Post-COVID-19 interdisciplinary studies, confirming that we are facing a truly intricate biomedicine story.

Che-1 modulates the decision between cell cycle arrest and apoptosis by its binding to p53.

The tumor suppressor p53 is mainly involved in the transcriptional regulation of a large number of growth-arrest- and apoptosis-related genes. However, a clear understanding of which factor/s influences the choice between these two opposing p53-dependent outcomes remains largely elusive. We have previously described that in response to DNA damage, the RNA polymerase II-binding protein Che-1/AATF transcriptionally activates p53. Here, we show that Che-1 binds directly to p53. This interaction essentially occurs in the first hours of DNA damage, whereas it is lost when cells undergo apoptosis in response to posttranscriptional modifications. Moreover, Che-1 sits in a ternary complex with p53 and the oncosuppressor Brca1. Accordingly, our analysis of genome-wide chromatin occupancy by p53 revealed that p53/Che1 interaction results in preferential transactivation of growth arrest p53 target genes over its pro-apoptotic target genes. Notably, exposure of Che-1(+/-) mice to ionizing radiations resulted in enhanced apoptosis of thymocytes, compared with WT mice. These results confirm Che-1 as an important regulator of p53 activity and suggest Che-1 to be a promising yet attractive drug target for cancer therapy.

Hippocampal dynamics of synaptic NF-kappa B during inhibitory avoidance long-term memory consolidation in mice.

Since the discovery that long-term memory is dependent on protein synthesis, several transcription factors have been found to participate in the transcriptional activity needed for its consolidation. Among them, NF-kappa B is a constitutive transcription factor whose nuclear activity has proven to be necessary for the consolidation of inhibitory avoidance in mice. This transcription factor has a wide distribution in the nervous system, with a well-reported presence in dendrites and synaptic terminals. Here we report changes in synaptosomal NF-kappa B localization and activity, during long-term memory consolidation. Activity comparison of synaptosomal and nuclear NF-kappa B, indicates different dynamics for both localizations. In this study we identify two pools of synaptosomal NF-kappa B, one obtained with the synaptoplasm (free fraction) and the second bound to the synaptosomal membranes. During the early steps of consolidation the first pool is activated, as the membrane associated transcription factor fraction increases and concomitantly the free fraction decreases. These results suggest that the activation of synaptic NF-kappa B and its translocation to membranes are part of the consolidation of long-term memory in mice.

HIPK2 sustains apoptotic response by phosphorylating Che-1/AATF and promoting its degradation.

Che-1/AATF is an RNA polymerase II-binding protein that is involved in the regulation of gene transcription, which undergoes stabilization and accumulation in response to DNA damage. We have previously demonstrated that following apoptotic induction, Che-1 protein levels are downregulated through its interaction with the E3 ligase HDM2, which leads to Che-1 degradation by ubiquitylation. This interaction is mediated by Pin1, which determines a phosphorylation-dependent conformational change. Here we demonstrate that HIPK2, a proapoptotic kinase, is involved in Che-1 degradation. HIPK2 interacts with Che-1 and, upon genotoxic stress, phosphorylates it at specific residues. This event strongly increases HDM2/Che-1 interaction and degradation of Che-1 protein via ubiquitin-dependent proteasomal system. In agreement with these findings, we found that HIPK2 depletion strongly decreases Che-1 ubiquitylation and degradation. Notably, Che-1 overexpression strongly counteracts HIPK2-induced apoptosis. Our results establish Che-1 as a new HIPK2 target and confirm its important role in the cellular response to DNA damage.

Che-1 activates XIAP expression in response to DNA damage.

X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis proteins family that selectively binds and inhibits caspase-3, -7 and -9. As such, XIAP is an extremely potent suppressor of apoptosis and an attractive target for cancer treatment. Che-1 is an antiapoptotic agent involved in the control of gene transcription and cell proliferation. Recently, we showed that the checkpoint kinases ATM/ATR and checkpoint kinase 2 physically and functionally interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce transcription of p53, and Che-1 depletion strongly sensitizes tumor cells to anticancer drugs. Here we show that Che-1 activates XIAP expression in response to DNA damage. This effect is mediated by Che-1 phosphorylation and requires NF-kappaB. Notably, we found that XIAP expression is necessary for antiapoptotic activity of Che-1 and that in vivo downregulation of Che-1 by small interference RNA strongly enhanced the cytotoxicity of anticancer drugs.

The artificial zinc finger protein 'Blues' binds the enhancer of the fibroblast growth factor 4 and represses transcription.

The design of novel genes encoding artificial transcription factors represents a powerful tool in biotechnology and medicine. We have engineered a new zinc finger-based transcription factor, named Blues, able to bind and possibly to modify the expression of fibroblast growth factor 4 (FGF-4, K-fgf), originally identified as an oncogene. Blues encodes a three zinc finger peptide and was constructed to target the 9 bp DNA sequence: 5'-GTT-TGG-ATG-3', internal to the murine FGF-4 enhancer, in proximity of Sox-2 and Oct-3 DNA binding sites. Our final aim is to generate a model system based on artificial zinc finger genes to study the biological role of FGF-4 during development and tumorigenesis.

The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

Identification of a novel partner of RNA polymerase II subunit 11, Che-1, which interacts with and affects the growth suppression function of Rb.

hRPB11 is a core subunit of RNA polymerase II (pol II) specifically down-regulated on doxorubicin (dox) treatment. Levels of this protein profoundly affect cell differentiation, cell proliferation, and tumorigenicity in vivo. Here we describe Che-1, a novel human protein that interacts with hRPB11. Che-1 possesses a domain of high homology with Escherichia coli RNA polymerase final sigma-factor 70 and SV40 large T antigen. In addition, we report that Che-1 interacts with the retinoblastoma susceptibility gene (Rb) by two distinct domains. Functionally, we demonstrate that Che-1 represses the growth suppression function of Rb, counteracting the inhibitory action of Rb on the trans-activation function of E2F1. These results identify a novel protein that binds Rb and the core of pol II, and suggest that Che-1 may be part of transcription regulatory complex.

The RNA polymerase II core subunit 11 interacts with keratin 19, a component of the intermediate filament proteins.

We have previously cloned the human RNA polymerase II subunit 11, as a doxorubicin sensitive gene product. We suggested multiple tasks for this subunit, including structural and regulatory roles. With the aim to clarify the human RNA polymerase II subunit 11 function, we have identified its interacting protein partners using the yeast two-hybrid system. Here, we show that human RNA polymerase II subunit 11 specifically binds keratin 19, a component of the intermediate filament protein family, which is expressed in a tissue and differentiation-specific manner. In particular, keratin 19 is a part of the nuclear matrix intermediate filaments. We provide evidence that human RNA polymerase II subunit 11 interacts with keratin 19 via its N-terminal alpha motif, the same motif necessary for its interaction with the human RNA polymerase II core subunit 3. We found that keratin 19 contains two putative leucine zipper domains sharing peculiar homology with the alpha motif of human RNA polymerase II subunit 3. Finally, we demonstrate that keratin 19 can compete for binding human RNA polymerase II subunit 11/human RNA polymerase II subunit 3 in vitro, suggesting a possible regulatory role for this molecule in RNA polymerase II assembly/activity.

The interacting RNA polymerase II subunits, hRPB11 and hRPB3, are coordinately expressed in adult human tissues and down-regulated by doxorubicin.

We previously isolated the human RPB11 cDNA, encoding the 13.3 kDa subunit of RNA polymerase II, and demonstrated that expression of this subunit is modulated by doxorubicin. Using hRPB11 as bait in a yeast two-hybrid system, two cDNA variants encoding a second RNA polymerase II subunit, hRPB3, have now been isolated and characterized. These two hRPB3 mRNA species differed in 3' UTR region length, the longer transcript containing the AU-rich sequence motif that mediates mRNA degradation. Both hRPB11 and hRPB3 transcripts share a similar pattern of distribution in human adult tissues, with particularly high levels in both heart and skeletal muscle, and the expression of both is down-regulated by doxorubicin as found previously for the hRPB11 subunit. Taken together, these findings suggest that the interaction between hRPB3 and hRPB11 is fundamental for their function and that this heterodimer is involved in doxorubicin toxicity.

Binding properties of the artificial zinc fingers coding gene Sint1.

On the basis of the recognition "code" that suggests specific rules between zinc finger's primary structure and the finger's potential DNA binding sites, we have constructed a new three-zinc finger coding gene to target the nine base pair DNA sequence: 5'-TGG-ATG-GAC-3'. This artificial gene named "Sint1" belongs to the Cys2-His2 zinc finger type. The amino acid positions, crucial for DNA binding, have been specifically chosen on the basis of the amino acid/base contacts more frequently represented in the available list of the proposed recognition "code". Here we demonstrate that Sint1 protein binds specifically the double strand "code" DNA target, with a dissociation constant (Kd) comparable to the Kd of the well known Zif268 protein. Sint1 "code" deduced and the "experimental" selected DNA binding sites share five nucleotide positions. Interestingly, Sint1 shows both high affinity and specificity toward the single strand "code" DNA binding site, with a Kd comparable to the corresponding double strand DNA target. Moreover, we prove that Sint1 is able to bind RNA similarly to several natural zinc finger proteins.

Synthesis of a new zinc finger peptide; comparison of its 'code' deduced and 'CASTing' derived binding sites.

Using two synthetic oligonucleotides, we have constructed a new gene containing three zinc finger motifs of the Cys2-His2 type. We named this artificial gene 'Mago'. The Mago nucleotide triplets encoding the amino acid positions, described to be crucial for DNA binding specificity, have been chosen on the basis of the proposed recognition 'code' that relates the zinc finger's primary structure to the DNA binding target. Here we demonstrate that Mago protein specifically binds the 'code' DNA target, with a dissociation constant (Kd) comparable to the Kd of the well known Zif268 protein with its binding site. Moreover, we show that the deduced Mago 'code' and the 'experimental' selected DNA binding sites are almost identical, differing only in two nucleotides at the side positions.

Double-stranded internucleosomal cleavage of apoptotic DNA is dependent on the degree of differentiation in muscle cells.

Apoptotic cell death has been correlated to DNA fragmentation into discrete segments corresponding to the length of nucleosomal protected fragments of 180-200 base pairs or multiples of it. This DNA degradation has been ascribed to endonuclease activity that cleaves internucleosomally, thus giving rise to a ladder distribution upon electrophoretic migration. This strict correlation was, however, shown to have notable exceptions, since in some cases only single strand cleavage in the internucleosomal DNA regions has been observed (Tomei, D. L., Shapiro, P. J., and Cope, O. F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 853-857). In the present work we show that mouse muscle cells, able to differentiate in vitro, if subjected to apoptosis present no DNA degradation into ladder form unless differentiation is previously induced. Furthermore, C3H/10T1/2 fibroblast cells, known to undergo apoptosis without DNA ladder formation, if converted to a myogenic program by MyoD expression, display internucleosomal DNA degradation upon induction of differentiation.

Zfp60, a mouse zinc finger gene expressed transiently during in vitro muscle differentiation.

The complete cDNA coding sequence of the zinc finger gene Zfp60 is reported. The predicted amino acid sequence of the Zfp60 protein has been found to contain 19 zinc finger motives clustered at the C-terminus. At its N-terminus, Zfp60 shares with other members of the zinc finger gene family two additional conserved amino acid modules named Kruppel Associated Boxes (KRAB). The expression patterns of Zfp60, MyoD and MHC mRNAs have been followed during in vitro myogenic differentiation of C2 cells. We show that the bacterial produced Zfp60 protein binds DNA only in presence of zinc ions. Zfp60 locus has been mapped in chromosome 7, where other Zfp loci are localised.

The product of Zfp59 (Mfg2), a mouse gene expressed at the spermatid stage of spermatogenesis, accumulates in spermatozoa nuclei.

We have described previously three mouse multifinger coding genes (C. Passananti et al., Proc. Natl. Acad. Sci. USA, 86: 9417-9421, 1989). We have analyzed the expression of one of them, termed Mfg2 and renamed Zfp59, and demonstrated that Zfp59 mRNA and its translation product are present in specific stages of mouse spermatogenesis. The predicted amino acid sequence of the Zfp59 protein has been found to consist of 16 zinc-finger motifs clustered at the COOH terminus and subdivided into two groups by a degenerate finger motif. At its NH2 terminus, Zfp59 shares with other members of the zinc finger gene family two additional conserved amino acid modules A and B, described as either FAX, KRAB, or FPB domains. By means of Northern blot, Western blot analysis, and immunohistochemical localization, Zfp59 mRNA and its translation product were shown to be synthesized specifically during the postmeiotic phase of male germ line differentiation. By immunoelectron microscopy, the Zfp59 protein has been shown to accumulate in the nuclei of mature sperms in association with the nuclear matrix.

A polyomavirus enhancer mutant confers ubiquitous high transcriptional efficiency to the SV40 late promoter.

To identify expression plasmids with high efficiency of transcription and with a broad tissue and cell range, we have constructed a recombinant vector combining the late SV40 promoter and the polyomavirus regulatory region derived from a mutant (PyNB11/1) which displays a very wide host range. We show that these recombinant enhancer-promoters are efficient drivers for heterologous gene transcription and expression in vitro in all mouse and human cells tested. The most active combination we identified contained the mutant enhancer (PyNB11/1) in the late orientation. This construct was able to promote a high efficiency of expression without significant fluctuation between cells of different tissutal origin or different differentiative stage. A possible interpretation of these results is discussed.

Mutations in the VP1 coding region of polyomavirus determine differentiating stage specificity.

Polyomavirus mutants capable of replicating in undifferentiated murine C2 myoblasts were selected and characterized. These mutants grow normally in 3T6 mouse fibroblast cells, and they do not complement the wild-type virus in coinfection experiments of C2 myoblasts. Of 12 isolates, 10 possess duplications of the regulatory region including the enhancer A domain. On the bases of the regulatory region structure and the presence and length of the enhancer duplication, the mutant viruses could be grouped into three classes. One mutant class (e.g., PyMB3) possesses an enhancer duplication of 91 bp identical to that of a previously characterized polyomavirus mutant, PyNB11/1. We have demonstrated that this enhancer duplication gives rise at its junction to a novel recognition motif for the transcriptional factor NF-1 (M. Caruso, C. Iacobini, C. Passananti, A. Felsani, and P. Amati, EMBO J. 9:947-955, 1990). The regulatory region PyMB3 virus recombined in a wild-type genome context maintains the mutant phenotype. The other two types of mutants, one with a 30-bp enhancer duplication (e.g., PyMB40) and one with a wild-type enhancer structure (e.g., PyMB27), possess two similar but distinct 6-bp deletions in the same region of the VP1 coding gene. In both cases, the ability to replicate in undifferentiated C2 myoblasts is strictly correlated to the mutation in the VP1 coding region.

Constitutive synthesis of polyoma antisense RNA renders cells immune to virus infection.

Mouse fibroblasts were stably transfected with expression plasmids in which sequences of the early region of polyomavirus were inserted both in sense and antisense orientation. The cell lines that synthesize in the antisense orientation, a 1195-bp viral genome fragment covering the Ori, Cap, ATG, and all of the early mRNA splicing sites acquire resistance to viral infection. Smaller fragments covering Ori, Cap, and ATG sites or the splicing sites, as well as fragments cloned in sense orientation, failed to confer cell immunity to polyoma infection. The resistance proved to be directly dependent upon the specific antisense RNA and to be inversely proportional to the multiplicity of infecting polyoma.

Protein recognition sites in polyomavirus enhancer: formation of a novel site for NF-1 factor in an enhancer mutant and characterization of a site in the enhancer D domain.

Polyomavirus mutants selected for modified host range exhibit DNA sequence alterations in the regulatory region, which consist mainly of duplications and/or deletions. Single base pair mutations have also been observed, which create or abolish DNA sequence motifs recognized by DNA-binding regulatory factors. The present work deals with the molecular characterization of a Polyoma mutant (PyNB11/1), selected for its high efficiency of growth in neuroblastoma cells. The enhancer region of PyNB11/1 displays a 91 bp tandem duplication harbouring a novel DNA sequence motif created at the boundary of the duplicated fragment. This motif is absent in the wild-type enhancer and is specifically recognized by a nuclear factor that belongs to the NF-1 family of transcription factors. We also report the characterization of an as yet unidentified DNA sequence motif in the D domain of the viral enhancer, that represents the binding site for a nuclear factor that is ubiquitous and comparably abundant in several murine cell types.

Mouse genes coding for "zinc-finger"-containing proteins: characterization and expression in differentiated cells.

By using the zinc-finger region of human cHF.10 cDNA as a probe at low-stringency hybridization conditions, several individual phages from a mouse skeletal muscle cDNA library have been isolated. The amino acid sequences of the "zinc-finger" domains derived from the DNA sequences of three cDNA clones are shown. The expression of the corresponding mRNAs in three cell lines (NIH 3T3, F9 teratocarcinoma, and C2 myoblast cells) at different stages of differentiation and in eight adult mouse tissues has been analyzed. The transcription of these genes is induced during the in vitro differentiation of the cell lines tested. These three genes are widely and evenly expressed in adult mouse tissues, with the remarkable exception of one that is expressed predominantly in the testis.