n1.  Oligomerization regulates the interaction of Gemin5 with members of the SMN complex and the translation machinery

Rosario Francisco-Velilla, Salvador Abellan, Azman Embarc-Buh & Encarnacion Martinez-Salas

RNA-binding proteins are multifunctional molecules impacting on multiple steps of gene regulation. Gemin5 was initially identified as a member of the survival of motor neurons (SMN) complex. The protein is organized in structural and functional domains, including a WD40 repeats domain at the N-terminal region, a tetratricopeptide repeat (TPR) dimerization module at the central region, and a non-canonical RNA-binding site at the C-terminal end. The TPR module allows the recruitment of the endogenous Gemin5 protein in living cells and the assembly of a dimer in vitro. However, the biological relevance of Gemin5 oligomerization is not known. Here we interrogated the Gemin5 interactome focusing on oligomerization-dependent or independent regions. We show that the interactors associated with oligomerization-proficient domains were primarily annotated to ribosome, splicing, translation regulation, SMN complex, and RNA stability. The presence of distinct Gemin5 protein regions in polysomes highlighted differences in translation regulation based on their oligomerization capacity. Furthermore, the association with native ribosomes and negative regulation of translation was strictly dependent on both the WD40 repeats domain and the TPR dimerization moiety, while binding with the majority of the interacting proteins, including SMN, Gemin2, and Gemin4, was determined by the dimerization module. The loss of oligomerization did not perturb the predominant cytoplasmic localization of Gemin5, reinforcing the cytoplasmic functions of this essential protein. Our work highlights a distinctive role of the Gemin5 domains for its functions in the interaction with members of the SMN complex, ribosome association, and RBP interactome.

Published: 28 June 2024    https://doi.org/10.1038/s41420-024-02057-5

n2.  Nucleolar stress caused by arginine-rich peptides triggers a ribosomopathy and accelerates aging in mice

Oleksandra Sirozh, Anabel Saez-Mas, Bomi Jung, Laura Sanchez-Burgos, Eduardo Zarzuela, Sara Rodrigo-Perez, Ivan Ventoso, Vanesa Lafarga, Oscar Fernandez-Capetillo

Nucleolar stress (NS) has been associated with age-related diseases such as cancer or neurodegeneration. To investigate how NS triggers toxicity, we used (PR)n arginine-rich peptides present in some neurodegenerative diseases as inducers of this perturbation. We here reveal that whereas (PR)n expression leads to a decrease in translation, this occurs concomitant with an accumulation of free ribosomal (r) proteins. Conversely, (PR)n-resistant cells have lower rates of r-protein synthesis, and targeting ribosome biogenesis by mTOR inhibition or MYC depletion alleviates (PR)n toxicity in vitro. In mice, systemic expression of (PR)97 drives widespread NS and accelerated aging, which is alleviated by rapamycin. Notably, the generalized accumulation of orphan r-proteins is a common outcome of chemical or genetic perturbations that induce NS. Together, our study presents a general model to explain how NS induces cellular toxicity and provides in vivo evidence supporting a role for NS as a driver of aging in mammals.

Keywords: nucleolar stress, aging, nucleolus, ribosomal proteins, ribosomopathy

Published: 18 April 2024    https://doi.org/10.1016/j.molcel.2024.02.031

n3.  A Proteogenomic Approach to Unravel New Proteins Encoded in the Leishmania donovani (HU3) Genome

Javier Adán-Jiménez, Alejandro Sánchez-Salvador, Esperanza Morato, Jose Carlos Solana, Begoña Aguado, and Jose M. Requena

Published: 13 June 202    https://doi.org/10.3390/genes15060775

n4. Sexual dimorphism in the antitumor immune responses elicited by the combination of fasting and chemotherapy

Andrés Pastor-Fernández, Manuel Montero Gómez de las Heras, Jose Ignacio Escrig-Larena, Marta Barradas, Cristina Pantoja, Adrian Plaza, Jose Luis Lopez-Aceituno, Esther Durán, Alejo Efeyan, Maria Mittelbrunn, Lola Martinez, Pablo Jose Fernandez-Marcos

The aim of our study was to elucidate the role of sex in the beneficial anti-tumoral effects of combining fasting and chemotherapy. For this, we inoculated B16-F10-derived melanoma allografts into immunocompetent male and female mice.

Published: 21 March 2024    https://doi.org/10.1002/cac2.12535

n5.  Phospholipid scramblase 1: an essential component of the nephrocyte slit diaphragm

Vicente Castillo-Mancho, Alexandra Atienza-Manuel, Jorge Sarmiento-Jiménez, Mar Ruiz-Gómez & Joaquim Culi

Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca²⁺-binding sites in Scramb1 are essential for its activity raising the possibility that Ca²⁺ signaling may control the assembly of SDs by impacting on Scramb1 activity.

Published:  15 June 2024    https://doi.org/10.1007/s00018-024-05287-z

n6.  Novel insights into the sexual dimorphism-associated immune response

José M. Izquierdo

TIn their recent article in Science, Chi et al. [1] characterized a mechanism that regulates tissue/organ-specific immunity in mice. Their findings revealed that the androgen type 2 innate lymphoid cell-dendritic cell axis played a prominent role in a microbiota-and sex hormone-dependent manner. These observations impli-cate sexual immune dimorphism as a molecular, cellular and tissue/organ-specific regulatory hub for the (im)balance between health and disease.

Published:  13 May 2024    https://doi.org/10.1038/s41423-024-01177-3

n7.  Cell proliferation and Notch signaling coordinate the formation of epithelial folds in the Drosophila leg

Alonso Rodríguez, David Foronda, Sergio Córdoba, Daniel Felipe-Cordero, Antonio Baonza, David G. Miguez, Carlos Estella

The formation of complex three-dimensional organs during development requires precise coordination between patterning networks and mechanical forces. In particular, tissue folding is a crucial process that relies on a combination of local and tissue-wide mechanical forces. Here, we investigate the contribution of cell proliferation to epithelial morphogenesis using the Drosophila leg tarsal folds as a model. We reveal that tissue-wide compression forces generated by cell proliferation, in coordination with the Notch signaling pathway, are essential for the formation of epithelial folds in precise locations along the proximo-distal axis of the leg. As cell numbers increase, compressive stresses arise, promoting the folding of the epithelium and reinforcing the apical constriction of invaginating cells. Additionally, the Notch target dysfusion plays a key function specifying the location of the folds, through the apical accumulation of F-actin and the apico-basal shortening of invaginating cells. These findings provide new insights into the intricate mechanisms involved in epithelial morphogenesis, highlighting the crucial role of tissue-wide forces in shaping a three-dimensional organ in a reproducible manner.

Key Words:  Drosophila, Notch, Cell proliferation, Epithelial folding, Morphogenesis, Tissue compression

Published: 16 April 2024    https://doi.org/10.1242/dev.202384

n8.  PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice

Ainhoa Martínez-Pizarro, Sara Picó, Arístides López-Márquez, Claudia Rodriguez-López, Elena Montalvo, Mar Alvarez, Margarita Castro, Santiago Ramón-Maiques, Belén Pérez, José J Lucas, Eva Richard, Lourdes R Desviat

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3′ splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

Key Words:  phenylketonuria, humanized mouse model, splice variant, CRISPR/Cas, autophagy

Published:  15 June 2024   https://doi.org/10.1093/hmg/ddae051

n9.  Human in vivo evidence of associations between herpes simplex virus and cerebral amyloid-beta load in normal aging

Jose L.Cantero, Mercedes Atienza, Isabel Sastre, María Jesús Bullido

Background: Mounting data suggests that herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of AD, possibly instigating amyloid-beta (Aβ) accumulation decades before the onset of clinical symptoms. However, human in vivo evidence linking HSV-1 infection to AD pathology is lacking in normal aging, which may contribute to the elucidation of the role of HSV-1 infection as a potential AD risk factor.

Methods: To shed light into this question, serum anti-HSV IgG levels were correlated with 18F-Florbetaben-PET binding to Aβ deposits and blood markers of neurodegeneration (pTau181 and neurofilament light chain) in cognitively normal older adults. Additionally, we investigated whether associations between anti-HSV IgG and AD markers were more evident in APOE4 carriers.

Results: We showed that increased anti-HSV IgG levels are associated with higher Aβ load in fronto-temporal regions of cognitively normal older adults. Remarkably, these cortical regions exhibited abnormal patterns of resting state-functional connectivity (rs-FC) only in those individuals showing the highest levels of anti-HSV IgG. We further found that positive relationships between anti-HSV IgG levels and Aβ load, particularly in the anterior cingulate cortex, are moderated by the APOE4 genotype, the strongest genetic risk factor for AD. Importantly, anti-HSV IgG levels were unrelated to either subclinical cognitive deficits or to blood markers of neurodegeneration.

Conclusions: All together, these results suggest that HSV infection is selectively related to cortical Aβ deposition in normal aging, supporting the inclusion of cognitively normal older adults in prospective trials of antimicrobial therapy aimed at decreasing the AD risk in the aging population.

Published: 03 April 2024    https://doi.org/10.1186/s13195-024-01437-4

n10. In vivo cyclic overexpression of Yamanaka factors restricted to neurons reverses age-associated phenotypes and enhances memory performance

Alejandro Antón-Fernández, Marta Roldán-Lázaro, Laura Vallés-Saiz, Jesús Ávila, Félix Hernández

In recent years, there has been success in partially reprogramming peripheral organ cells using cyclic Yamanaka transcription factor (YF) expression, resulting in the reversal of age-related pathologies. In the case of the brain, the effects of partial reprogramming are scarcely known, and only some of its effects have been observed through the widespread expression of YF. This study is the first to exclusively partially reprogram a specific subpopulation of neurons in the cerebral cortex of aged mice. The in vivo model demonstrate that YF expression in postmitotic neurons does not dedifferentiate them, and it avoids deleterious effects observed with YF expression in other cell types. Additionally, our study demonstrates that only cyclic, not continuous, expression of YF result in a noteworthy enhancement of cognitive function in adult mice. This enhancement is closely tied to increased neuronal activation in regions related to memory processes, reversed aging-related epigenetic markers and to increased plasticity, induced by the reorganization of the extracellular matrix. These findings support the therapeutic potential of targeted partial reprogramming of neurons in addressing age-associated phenotypes and neurodegenerative diseases correlated with aging.

Published: 24 May 2024    https://doi.org/10.1038/s42003-024-06328-w

n11.  Early heart and skeletal muscle mitochondrial response to a moderate hypobaric hypoxia environment

Jerónimo Aragón-Vela, Rafael A. Casuso, Ana Sagrera Aparisi, Julio Plaza-Díaz, Ascensión Rueda-Robles, Agustín Hidalgo-Gutiérrez, Luis Carlos López, Andrea Rodríguez-Carrillo, José Antonio Enriquez, Sara Cogliati, Jesús R. Huertas

In eukaryotic cells, aerobic energy is produced by mitochondria through oxygen uptake. However, little is known about the early mitochondrial responses to moderate hypobaric hypoxia (MHH) in highly metabolic active tissues. Here, we describe the mitochondrial responses to acute MHH in the heart and skeletal muscle. Rats were randomly allocated into a normoxia control group (n = 10) and a hypoxia group (n = 30), divided into three groups (0, 6, and 24 h post-MHH). The normoxia situation was recapitulated at the University of Granada, at 662 m above sea level. The MHH situation was performed at the High-Performance Altitude Training Centre of Sierra Nevada located in Granada at 2320 m above sea level. We found a significant increase in mitochondrial supercomplex assembly in the heart as soon as the animals reached 2320 m above sea level and their levels are maintained 24 h post-exposure, but not in skeletal muscle. Furthermore, in skeletal muscle, at 0 and 6 h, there was increased dynamin-related protein 1 (Drp1) expression and a significant reduction in Mitofusin 2. In conclusion, mitochondria from the muscle and heart respond differently to MHH: mitochondrial supercomplexes increase in the heart, whereas, in skeletal muscle, the mitochondrial pro-fission response is trigged. Considering that skeletal muscle was not actively involved in the ascent when the heart was beating faster to compensate for the hypobaric, hypoxic conditions, we speculate that the different responses to MHH are a result of the different energetic requirements of the tissues upon MHH.

Published: 17 April 2024    https://doi.org/10.1113/JP285516

n12.  Poxvirus Immune Evasion

Bruno Hernaez, and Antonio Alcamí

Poxviruses have evolved a wide array of mechanisms to evade the immune response, and we provide an overview of the different immunomodulatory strategies. Poxviruses prevent the recognition of viral DNA that triggers the immune responses and inhibit signaling pathways within the infected cell. A unique feature of poxviruses is the production of secreted proteins that mimic cytokines and cytokine receptors, acting as decoy receptors to neutralize the activity of cytokines and chemokines. The capacity of these proteins to evade cellular immune responses by inhibiting cytokine activation is complemented by poxviruses’ strategies to block natural killer cells and cytotoxic T cells, often through interfering with antigen presentation pathways. Mechanisms that target complement activation are also encoded by poxviruses. Virus-encoded proteins that target immune molecules and pathways play a major role in immune modulation, and their contribution to viral pathogenesis, facilitating virus replication or preventing immunopathology, is discussed.

Key Words:  poxvirus, immune pathway, cytokine, decoy receptor, cell-mediated immunity, viral pathogenesis

Published: June 2024    https://doi.org/10.1146/annurev-immunol-090222-110227

n13.  Thermostable in vitro transcription-translation compatible with microfluidic droplets

Ana L. J. L. Ribeiro, Patricia Pérez-Arnaiz, Mercedes Sánchez-Costa, Lara Pérez, Marcos Almendros, Liisa Vliet, Fabrice Gielen, Jesmine Lim, Simon Charnock, Florian Hollfelder, J. Eduardo González-Pastor, José Berenguer, Aurelio Hidalgo

Background; In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures.

Objectives: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.

Results: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time.

Conclusions: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Published: 10 June 2024    https://doi.org/10.1186/s12934-024-02440-y

n14.  Association of Acidotolerant Cyanobacteria to Microbial Mats below pH 1 in Acidic Mineral Precipitates in Río Tinto River in Spain

Felipe Gómez, Nuria Rodríguez, José Antonio Rodríguez-Manfredi, Cristina Escudero, Ignacio Carrasco-Ropero, José M. Martínez, Marco Ferrari, Simone De Angelis, Alessandro Frigeri, Maite Fernández-Sampedro, Ricardo Amils

This report describes acidic microbial mats containing cyanobacteria that are strongly associated to precipitated minerals in the source area of Río Tinto. Río Tinto (Huelva, Southwestern Spain) is an extreme acidic environment where iron and sulfur cycles play a fundamental role in sustaining the extremely low pH and the high concentration of heavy metals, while maintaining a high level of microbial diversity. These multi-layered mineral deposits are stable all year round and are characterized by a succession of thick greenish-blue and brownish layers mainly composed of natrojarosite. The temperature and absorbance above and below the mineral precipitates were followed and stable conditions were detected inside the mineral precipitates. Different methodologies, scanning and transmission electron microscopy, immunological detection, fluorescence in situ hybridization, and metagenomic analysis were used to describe the biodiversity existing in these microbial mats, demonstrating, for the first time, the existence of acid-tolerant cyanobacteria in a hyperacidic environment of below pH 1. Up to 0.46% of the classified sequences belong to cyanobacterial microorganisms, and 1.47% of the aligned DNA reads belong to the Cyanobacteria clade.

Key Words:  extremophiles; acidophiles; cyanobacteria; natrojarosite; endolithic ecosystems; earth analogues; astrobiology

Published: 19 April 2024    https://doi.org/10.3390/microorganisms12040829

n15.  Differential requirement for RecFOR pathway components in Thermus thermophilusXXX

Cristina L. Gómez-Campo, Ali Abdelmoteleb, Verónica Pulido, Marc Gost, Dione L. Sánchez-Hevia, José Berenguer, Mario Mencía

Recombinational repair is an important mechanism that allows DNA replication to overcome damaged templates, so the DNA is duplicated timely and correctly. The RecFOR pathway is one of the common ways to load RecA, while the RuvABC complex operates in the resolution of DNA intermediates. We have generated deletions of recO, recR and ruvB genes in Thermus thermophilus, while a recF null mutant could not be obtained. The recO deletion was in all cases accompanied by spontaneous loss of function mutations in addA or addB genes, which encode a helicase-exonuclease also key for recombination. The mutants were moderately affected in viability and chromosome segregation. When we generated these mutations in a Δppol/addAB strain, we observed that the transformation efficiency was maintained at the typical level of Δppol/addAB, which is 100-fold higher than that of the wild type. Most mutants showed increased filamentation phenotypes, especially ruvB, which also had DNA repair defects. These results suggest that in T. thermophilus (i) the components of the RecFOR pathway have differential roles, (ii) there is an epistatic relationship of the AddAB complex over the RecFOR pathway and (iii) that neither of the two pathways or their combination is strictly required for viability although they are necessary for normal DNA repair and chromosome segregation.

Published: 01 June 2024    https://doi.org/10.1111/1758-2229.13269