Ana Sanz

Functional and genomic analyses of blocked protein O‐mannosylation in baker's yeast

O‐mannosylation is a crucial protein modification in eukaryotes that is initiated by the essential family of protein O‐mannosyltransferases (PMTs). Here we demonstrate that in the model yeast Saccharomyces cerevisiae rhodanine‐3‐acetic acid derivatives affect members of all PMT subfamilies. Specifically, we used OGT2468 to analyse genome‐wide transcriptional changes in response to general inhibition of O‐mannosylation in bakers yeast. PMT inhibition results in the activation of the cell wall integrity (CWI) pathway. Coinciding, the mitogen‐activated kinase Slt2p is activated in vivo and CWI pathway mutants are hypersensitive towards OGT2468. Further, induction of many target genes of the unfolded protein response (UPR) and ER‐associated protein degradation (ERAD) is observed. The interdependence of O‐mannosylation and UPR/ERAD is confirmed by genetic interactions between HAC1 and PMTs, and increased degradation of the ERAD substrate Pdr5p* in pmtΔ mutants. Transcriptome analyses further suggested that mating and filamentous growth are repressed upon PMT inhibition. Accordingly, in vivo mating efficiency and invasive growth are considerably decreased upon OGT2468 treatment. Quantitative PCR and ChIP analyses suggest that downregulation of mating genes is dependent on the transcription factor Ste12p. Finally, inhibitor studies identified a role of the Ste12p‐dependent vegetative signalling cascade in the adaptive response to inhibition of O‐mannosylation.

Rlm1 mediates positive autoregulatory transcriptional feedback that is essential for Slt2-dependent gene expression.

ABSTRACT Activation of the yeast cell wall integrity (CWI) pathway induces an adaptive transcriptional programme that is largely dependent on the transcription factor Rlm1 and the mitogen-activated protein kinase (MAPK) Slt2. Upon cell wall stress, the transcription factor Rlm1 is recruited to the promoters of RLM1 and SLT2, and exerts positive-feedback mechanisms on the expression of both genes. Activation of the MAPK Slt2 by cell wall stress is not impaired in strains with individual blockade of any of the two feedback pathways. Abrogation of the autoregulatory feedback mechanism on RLM1 severely affects the transcriptional response elicited by activation of the CWI pathway. In contrast, a positive trans-acting feedback mechanism exerted by Rlm1 on SLT2 also regulates CWI output responses but to a lesser extent. Therefore, a complete CWI transcriptional response requires not only phosphorylation of Rlm1 by Slt2 but also concurrent SLT2- and RLM1-mediated positive-feedback mechanisms; sustained patterns of gene expression are mainly achieved by positive autoregulatory circuits based on the transcriptional activation of Rlm1. Summary: Sustaining patterns of gene expression through the yeast CWI pathway are mainly achieved by positive Rlm1 autoregulatory circuits, which are essential for amplification of the transcriptional response.

Cooperation between SAGA and SWI/SNF complexes is required for efficient transcriptional responses regulated by the yeast MAPK Slt2

The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway.

'Strengthening the fungal cell wall through chitin-glucan cross-links: effects on morphogenesis and cell integrity'.

The cross‐linking of polysaccharides to assemble new cell wall in fungi requires transglycosylation mechanisms by which preexisting glycosidic linkages are broken and new linkages are created between the polysaccharides. The molecular mechanisms for these processes, which are essential for fungal cell biology, are only now beginning to be elucidated. Recent development of in vivo and in vitro biochemical approaches has allowed characterization of important aspects about the formation of chitin–glucan covalent cell wall cross‐links by cell wall transglycosylases of the CRH family and their biological function. Covalent linkages between chitin and glucan mediated by Crh proteins control morphogenesis and also play important roles in the remodeling of the fungal cell wall as part of the compensatory responses necessary to counterbalance cell wall stress. These enzymes are encoded by multigene families of redundant proteins very well conserved in fungal genomes but absent in mammalian cells. Understanding the molecular basis of fungal adaptation to cell wall stress through these and other cell wall remodeling enzymatic activities offers an opportunity to explore novel antifungal treatments and to identify potential fungal virulence factors.

Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases

Covalent cross‐links between chitin and glucan at the yeast cell wall are created by the transglycosylase activity of redundant proteins Crh1 and Crh2, with cleavage of β‐1,4 linkages of the chitin backbone and transfer of the generated molecule containing newly created reducing end onto the glucan acceptor. A three‐dimensional structure of Crh1 was generated by homology modeling based on the crystal structure of bacterial 1,3‐1,4‐β‐d‐glucanase, followed by site‐directed mutagenesis to obtain molecular insights into how these enzymes achieve catalysis. The residues of both proteins that are involved in their catalytic and binding activities have been characterized by measuring the ability of yeast cells expressing different versions of these proteins to transglycosylate oligosaccharides derived from β‐1,3‐glucan, β‐1,6‐glucan and chitin to the chitin at the cell wall. Within the catalytic site, residues E134 and E138 of Crh1, as well as E166 and E170 of Crh2, corresponding to the nucleophile and general acid/base, and also the auxiliary D136 and D168 of Crh1 and Crh2, respectively, are shown to be essential for catalysis. Mutations of aromatic residues F152, Y160 and W219, located within the carbohydrate‐binding cleft of the Crh1 model, also affect the transglycosylase activity. Unlike Crh1, Crh2 contains a putative carbohydrate‐binding module (CBM18) of unknown function. Modeling and functional analysis of site‐directed mutant residues of this CBM identified essential amino acids for protein folding and stability, as well as residues that tune the catalytic activity of Crh2.

Out of the TWEAKlight: Elucidating the Role of Fn14 and TWEAK in Acute Kidney Injury

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a tumor necrosis factor superfamily cytokine that activates the fibroblast growth factor-inducible-14 (Fn14) receptor. Functional studies have established a role of TWEAK/Fn14 in experimental acute kidney injury (AKI) and the AKI to chronic kidney disease transition through actions on tubular cells and renal fibroblasts. The renal cell expression of TWEAK and Fn14 is increased in human and experimental AKI and targeting TWEAK or Fn14 by genetic means or neutralizing antibodies was protective in kidney injury induced by folic acid overdose, ischemia-reperfusion, or unilateral ureteral obstruction. TWEAK/Fn14 targeting preserved renal function, and reduced tubular cell injury and death, nuclear factor-κB activation, chemokine expression, inflammatory cell infiltration by macrophages and T cells, myofibroblast numbers, and extracellular matrix deposition, while preserving the expression of the anti-aging factor klotho and the mitochondrial regulator Peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC1α), as well as of PGC1α-dependent genes. The beneficial in vivo effects of TWEAK/Fn14 targeting are consistent with known actions of TWEAK on kidney cells. We review the literature on TWEAK and AKI and propose further avenues of research to unravel the contribution of TWEAK to kidney injury. Although a randomized clinical trial of neutralizing anti-TWEAK antibodies for lupus nephritis recently was terminated for futility, AKI represents a potential target for clinical development because it is potentially lethal and, as opposed to severe lupus nephritis, is very common, lacks effective therapy, and is not autoimmune in nature.

rTMS stimulation to induce plastic changes at the language motor area in a patient with a left recidivant brain tumor affecting Broca's area

Introduction. Extent of resection is one of the most powerful predictors of outcome in surgery of gliomas. Tumors located within areas governing eloquence may impede a total tumor resection. Functional plasticity may be induced by therapeutic means, such as cortical stimulation with repetitive transcranial magnetic stimulation (rTMS). Thus, rTMS is a potential tool to induce an improvement of functions of eloquence menaced by brain tumors.  Material and Methods.We report a case of a 59-year-old woman operated for a left sided precentral oligodendroglioma with awake intraoperative stimulation, whose tumor could not be completely removed because it affected areas governing language. Nine months later the tumor progressed and the motor language functions worsened. We submitted the patient to rTMS directed to Brocas area, next to the anterior pole of the tumor, with the aim of improving motor language function before a new tumor resection attempt. We performed 12 daily sessions of theta-burst rTMS followed by intensive language rehabilitation for 10 minutes, and 5 different aspects of language were measured before, immediately after and 10 minutes after each session. Results.Repetition and nomination worsened immediately after each rTMS session, and improved after 10 min of rehabilitation. However, basal values improved globally along the experiment. Also, the impact of rTMS on motor language was increasingly less along the procedure. Conclusions.rTMS induces changes in Brocas area and this effect can be potentially used to improve language function in tumors located at or close to eloquent cortical areas .

The CWI Pathway: Regulation of the Transcriptional Adaptive Response to Cell Wall Stress in Yeast

Fungi are surrounded by an essential structure, the cell wall, which not only confers cell shape but also protects cells from environmental stress. As a consequence, yeast cells growing under cell wall damage conditions elicit rescue mechanisms to provide maintenance of cellular integrity and fungal survival. Through transcriptional reprogramming, yeast modulate the expression of genes important for cell wall biogenesis and remodeling, metabolism and energy generation, morphogenesis, signal transduction and stress. The yeast cell wall integrity (CWI) pathway, which is very well conserved in other fungi, is the key pathway for the regulation of this adaptive response. In this review, we summarize the current knowledge of the yeast transcriptional program elicited to counterbalance cell wall stress situations, the role of the CWI pathway in the regulation of this program and the importance of the transcriptional input received by other pathways. Modulation of this adaptive response through the CWI pathway by positive and negative transcriptional feedbacks is also discussed. Since all these regulatory mechanisms are well conserved in pathogenic fungi, improving our knowledge about them will have an impact in the developing of new antifungal therapies.

Targeting inflammation in diabetic nephropathy: a tale of hope

ABSTRACT Introduction: Diabetic nephropathy (DN) is the leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD). Beyond the new anti-diabetic drugs that possess markedly cardiovascular and renal protective effects, no novel direct therapies for DN have become available on the market in the last twenty years. Recently well-designed clinical trials for the treatment of DN, with attractive pathogenetic rationale, e.g. bardoxolone and atrasentan, were canceled or stopped because of safety concerns or lack of reaching the end points, respectively. Areas covered: In this review, we focus on the involvement of inflammation in the pathogenesis of DN. We update information from recent experimental and clinical studies that reported beneficial effects of several agents targeting chemokines, cytokines, transcription factors and kinases as well as several compounds with anti-inflammatory properties on DN. Expert opinion: Inflammation plays a key role in the DN progression. Preclinical studies have identified several anti-inflammatory molecules that effective decrease albuminuria and/or proteinuria. However, limited clinical trials in humans have been performed to confirm these results. Inhibitors of CCL2/CCR2, IL-1β and JAK/STAT pathways, and Nrf2 inducers are promising therapeutic options to improve the renal outcome of patients with DN, but appropriate clinical trials are necessary.

Slt2 MAPK association with chromatin is required for transcriptional activation of Rlm1 dependent genes upon cell wall stress

The regulation of gene expression through the cell wall integrity (CWI) pathway in yeast is mainly coordinated by the MAPK Slt2 and the transcription factor Rlm1. In this work, we elucidate a new role for Slt2 as a part of the transcriptional activation machinery that regulates CWI gene expression in response to cell wall stress. We show that Slt2 is recruited to promoters and coding regions of CWI Rlm1-dependent genes in response to stress. This phenomenon is dependent both on the activation of the MAPK and its kinase activity. Slt2 binding is also dependent on Rlm1 and SWI/SNF and SAGA complexes. During the initial steps of transcription, the catalytic activity of Slt2 on Rlm1 is critical for the binding of the activator to promoters in response to stress. In addition, Slt2 itself acts as a transactivator, as it is able to induce the transcription of CWI responsive genes when it is bound to promoters through the Rlm1 binding domain independently of its catalytic activity. Slt2 interacts with RNA Pol II in a Rlm1-dependent manner to provide further support to a role of this MAPK as an integral component of the transcriptional complexes under cell wall stress. Selective recruitment and progression of the complex Slt2-RNA Pol II from the promoters to the coding regions of Rlm1-dependent genes does not rely on Paf1, suggesting a different mechanism from that which is exerted by Slt2 on the Swi4/Swi6 (SBF)-regulated genes.