Nature-inspired peptide of MtDef4 C-terminus tail enables protein delivery in mammalian cells.

Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide's ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications.

Kidney-specific methylation patterns correlate with kidney function and are lost upon kidney disease progression.

BACKGROUND: Chronological and biological age correlate with DNA methylation levels at specific sites in the genome. Linear combinations of multiple methylation sites, termed epigenetic clocks, can inform us the chronological age and predict multiple health-related outcomes. However, why some sites correlating with lifespan, healthspan, or specific medical conditions remain poorly understood. Kidney fibrosis is the common pathway for chronic kidney disease, which affects 10% of European and US populations. RESULTS: Here we identify epigenetic clocks and methylation sites that correlate with kidney function. Moreover, we identify methylation sites that have a unique methylation signature in the kidney. Methylation levels in majority of these sites correlate with kidney state and function. When kidney function deteriorates, all of these sites regress toward the common methylation pattern observed in other tissues. Interestingly, while the majority of sites are less methylated in the kidney and become more methylated with loss of function, a fraction of the sites are highly methylated in the kidney and become less methylated when kidney function declines. These methylation sites are enriched for specific transcription-factor binding sites. In a large subset of sites, changes in methylation patterns are accompanied by changes in gene expression in kidneys of chronic kidney disease patients. CONCLUSIONS: These results support the information theory of aging, and the hypothesis that the unique tissue identity, as captured by methylation patterns, is lost as tissue function declines. However, this information loss is not random, but guided toward a baseline that is dependent on the genomic loci. SIGNIFICANCE STATEMENT: DNA methylation at specific sites accurately reflects chronological and biological age. We identify sites that have a unique methylation pattern in the kidney. Methylation levels in the majority of these sites correlate with kidney state and function. Moreover, when kidney function deteriorates, all of these sites regress toward the common methylation pattern observed in other tissues. Thus, the unique methylation signature of the kidney is degraded, and epigenetic information is lost, when kidney disease progresses. These methylation sites are enriched for specific and methylation-sensitive transcription-factor binding sites, and associated genes show disease-dependent changes in expression. These results support the information theory of aging, and the hypothesis that the unique tissue identity, as captured by methylation patterns, is lost as tissue function declines.

Immunorecognition of Streptococcus mutans secreted proteins protects against caries by limiting tooth adhesion.

INTRODUCTION: Childhood caries, a prevalent chronic disease, affects 60-90 % of children in industrialized regions, leading to lesions in both primary and permanent teeth. This condition precipitates hospital admissions, emergency room visits, elevated treatment costs, and missed school days, thereby impeding the child's academic engagement and increasing the likelihood of caries into adulthood. Despite multiple identified risk factors, significant interpersonal variability remains unexplained. The immune system generates a unique antibody repertoire, essential for maintaining a balanced and healthy oral microbiome. Streptococcus mutans is a primary contributor to the development of caries. METHODS: Employing mass spectrometry, we investigated the S. mutans proteins targeted by antibodies in children both with and without caries, delineating a fundamental suite of proteins discernible by the immune systems of a majority of individuals. Notably, this suite was enriched with proteins pivotal for bacterial adhesion. To ascertain the physiological implications of these discoveries, we evaluated the efficacy of saliva in thwarting S. mutans adherence to dental surfaces. RESULTS: Antibodies in most children recognized a core set of ten S. mutans proteins, with additional proteins identified in some individuals. There was no significant difference in the proteins identified by children with or without caries, but there was variation in antibody binding intensity to some proteins. Functionally, saliva from caries-free individuals, but not children with caries, was found to hinder the binding of S. mutans to teeth. These findings delineate the S. mutans proteome targeted by the immune system and suggest that the inhibition of bacterial adherence to teeth is a primary mechanism employed by the immune system to maintain oral balance and prevent caries formation. CONCLUSIONS: These findings enhance our knowledge of the immune system's function in oral health maintenance and caries prevention, shedding light on how immunoglobulins interact with S. mutans proteins. CLINICAL SIGNIFICANCE: Targeting S. mutans proteins implicated in bacterial adhesion could be a promising strategy for preventing childhood caries.

A novel computationally engineered collagenase reduces the force required for tooth extraction in an ex-situ porcine jaw model.

The currently employed tooth extraction methods in dentistry involve mechanical disruption of the periodontal ligament fibers, leading to inevitable trauma to the bundle bone comprising the socket walls. In our previous work, we have shown that a recombinantly expressed truncated version of clostridial collagenase G (ColG) purified from Escherichia coli efficiently reduced the force needed for tooth extraction in an ex-situ porcine jaw model, when injected into the periodontal ligament. Considering that enhanced thermostability often leads to higher enzymatic activity and to set the basis for additional rounds of optimization, we used a computational protein design approach to generate an enzyme to be more thermostable while conserving the key catalytic residues. This process generated a novel collagenase (ColG-variant) harboring sixteen mutations compared to ColG, with a nearly 4℃ increase in melting temperature. Herein, we explored the potential of ColG-variant to further decrease the physical effort required for tooth delivery using our established ex-situ porcine jaw model. An average reduction of 11% was recorded in the force applied to extract roots of mandibular split first and second premolar teeth treated with ColG-variant, relative to those treated with ColG. Our results show for the first time the potential of engineering enzyme properties for dental medicine and further contribute to minimally invasive tooth extraction.

Enhancement of Collagen-I Levels in Human Gingival Fibroblasts by Small Molecule Activation of HIF-1α.

Collagen is the most abundant protein in various mammalian tissues and has an essential role in various cellular processes. Collagen is necessary for food-related biotechnological applications such as cultivated meat, medical engineering, and cosmetics. High-yield expression of natural collagen from mammalian cells is challenging and not cost-effective. Thus, external collagen is obtained primarily from animal tissues. Under cellular hypoxia, overactivation of the transcription factor hypoxia-inducible factor (HIF) was shown to correlate with enhanced accumulation of collagen. Herein, we showed that the small molecule ML228, a known molecular activator of HIF, enhances the accumulation of collagen type-I in human fibroblast cells. We report an increase in collagen levels by 2.33 ± 0.33 when fibroblasts were incubated with 5 µM of ML228. Our experimental results demonstrated, for the first time, that external modulation of the hypoxia biological pathway can boost collagen levels in mammalian cells. Our findings pave the way for enhancing natural collagen production in mammals by altering cellular signaling pathways.

Single-cell transcriptomic analysis of oral masticatory and lining mucosa-derived mesenchymal stromal cells.

AIM: To reveal the heterogeneity of ex vivo-cultured human mesenchymal stromal cells derived from either masticatory or lining oral mucosa. MATERIALS AND METHODS: Cells were retrieved from the lamina propria of the hard palate and alveolar mucosa of three individuals. The analysis of transcriptomic-level differences was accomplished using single-cell RNA sequencing. RESULTS: Cluster analysis clearly distinguished between cells from the masticatory and lining oral mucosa, and revealed 11 distinct cell sub-populations, annotated as fibroblasts, smooth muscle cells or mesenchymal stem cells. Interestingly, cells presenting a mesenchymal stem cell-like gene expression pattern were predominantly found in masticatory mucosa. Although cells of masticatory mucosa origin were highly enriched for biological processes associated with wound healing, those from the lining oral mucosa were highly enriched for biological processes associated with the regulation of epithelial cells. CONCLUSIONS: Our previous work had shown that cells from the lining and masticatory oral mucosae are phenotypically heterogeneous. Here, we extend these findings to show that these changes are not the result of differences in averages but rather represent two distinct cell populations, with mesenchymal stem cells more common in masticatory mucosa. These features may contribute to specific physiological functions and have relevance for potential therapeutic interventions.

The Hidden Secrets of the Dental Calculus: Calibration of a Mass Spectrometry Protocol for Dental Calculus Protein Analysis.

Dental calculus is a solid deposit that forms and accumulates on the tooth surface, entrapping oral microorganisms, biomolecules, and other micro-debris found in the oral cavity. A mass spectrometry analysis of its protein content opens a vista into the subject's diet, oral flora, and even some aspects of health, thus providing new insight and expanding our knowledge of archaic cultures. Multiple experimental protocols have been proposed for the optimal extraction of proteins from dental calculus. Herein, we compared various experimental conditions in order to calibrate and validate a protocol for protein extraction. Our results show that a high concentration of acetic acid followed by mechanical crushing and sonication provided the highest protein yield, while acetone precipitation enabled the identification of more distinct proteins. We validated this protocol using archeological samples, identifying human and microbial proteins in specimens from the eighth and seventeenth centuries (approximately 250-1300 years ago). These findings demonstrate that the developed protocol is useful for studying excavated archaeological samples and that it might be utilized to explore the biohistory, dietary habits, and microbiome of archaic populations.

Fibroblasts from the oral masticatory and lining mucosa have different gene expression profiles and proliferation rates.

AIMS: To compare the gene expression profiles and proliferation rates of fibroblasts from the oral lining and masticatory mucosae. MATERIALS AND METHODS: Primary human fibroblasts were retrieved from the posterior masticatory hard palate and the lining alveolar mucosa of five individuals. The gene expression profile was evaluated using total RNA sequencing. The proliferation rate was determined colorimetrically. RESULTS: Substantial differences in specific gene groups and pathways were observed between fibroblasts from the two tissues. Significantly enriched gene ontology processes were focused on the extracellular components. Lining mucosa fibroblasts exhibited significantly higher expression of the principal structural collagens, cranial neural crest markers, and homeobox genes associated with positional memory. Masticatory mucosa fibroblasts showed greater expression of genes related to transforming growth factor-ß signalling, which may be associated with fibrosis. In addition, they expressed higher levels of the EP2 prostaglandin E2 receptor and Toll-like receptor 1. Finally, masticatory mucosa fibroblasts exhibited a 10%-30% higher proliferation rate. CONCLUSIONS: Fibroblasts from the lining and masticatory oral mucosae are phenotypically heterogeneous, presenting distinct gene expression profiles and proliferation rates. These features may contribute to their specific physiological functions and have relevance for potential therapeutic applications.

Lamin regulates the dietary restriction response via the mTOR pathway in Caenorhabditis elegans.

Animals subjected to dietary restriction (DR) have reduced body size, low fecundity, slower development, lower fat content and longer life span. We identified lamin as a regulator of multiple dietary restriction phenotypes. Downregulation of lmn-1, the single Caenorhabditis elegans lamin gene, increased animal size and fat content specifically in DR animals. The LMN-1 protein acts in the mTOR pathway, upstream of RAPTOR and S6 kinase ß1 (S6K), a key component of and target of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), respectively. DR excludes the mTORC1 activator RAGC-1 from the nucleus. Downregulation of lmn-1 restores RAGC-1 to the nucleus, a necessary step for the activation of the mTOR pathway. These findings further link lamin to metabolic regulation.

Exploring the nuclear lamina in health and pathology using C. elegans.

The eukaryotic genome inside the nucleus is enveloped by two membranes, the Outer Nuclear Membrane (ONM) and the Inner Nuclear Membrane (INM). Tethered to the INM is the nuclear lamina, a fibrillar network composed of lamins-the nuclear intermediate filaments, and membrane associated proteins. The nuclear lamina interacts with several nuclear structures, including chromatin. As most nuclear functions, including regulation of gene expression, chromosome segregation and duplication as well as nuclear structure, are highly conserved in metazoans, the Caenorhabditis elegans nematode serves as a powerful model organism to study nuclear processes and architecture. This translucent organism can easily be observed under a microscope as a live embryo, larvae and even adult. Here we will review the data on nuclear lamina composition and functions gathered from studies using C. elegans model organisms: We will discuss genome spatial organization and its contribution to gene expression. We will review both the interaction between the cytoplasm and the nucleus and mechanotransduction mechanism. Finally, we will discuss disease causing mutation in nuclear lamins, including the use of this animal model in diseases research.

Antibody-Driven Proximity Labeling in Fixed Tissues.

Protein function often depends on assemblies and interactions. These show complex spatial and temporal organization within the cell. Analysis of protein function can be greatly assisted by grouping proteins with their neighbors. Rather than relying on affinity, proximity labeling targets proteins proximal to the target of interest. We describe a protocol for antibody-guided deposition of tags in fixed and permeabilized cell lines and primary human tissue samples.

Small GTPases in C. elegans metabolism.

The mechanistic target of rapamycin (mTOR) is an evolutionary conserved protein with a serine/threonine kinase activity that regulates cell growth, proliferation, motility, survival, protein synthesis, autophagy and transcription. It is embedded in 2 large protein complexes: mTORC1 and mTORC2. Regulation of specific mTOR pathway functions depends on multiple GTPases, that act either as regulators of mTOR protein complexes, coupling energy availability with mTORC1 activity, or as downstream effectors of both mTORC1 and mTORC2. In this commentary, we highlight the advantages of studying the mTOR pathway in C. elegans, including the subcellular localization of the signaling pathway components and the animal phenotypes following tissue specific protein over-expression or knockdown. One important regulator that is not limited to the mTOR pathway is RHEB. We discuss in vitro and in vivo data suggesting that RHEB can function as an inhibitor of mTOR when not bound to GTP. RHEB-1 itself is regulated by Rab GDP dissociation inhibitor ß, which directly binds to ATX-2. We also highlight the roles of these proteins in dietary restriction-depended reduction in animal size and fat content.

Biotinylation by antibody recognition-a method for proximity labeling.

The high-throughput detection of organelle composition and proteomic mapping of protein environment directly from primary tissue as well as the identification of interactors of insoluble proteins that form higher-order structures have remained challenges in biological research. We report a proximity-based labeling approach that uses an antibody to a target antigen to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues, which allows proteins in close proximity to the target antigen to be captured and identified by mass spectrometry. We demonstrated the specificity and sensitivity of our method by examining the well-studied mitochondrial matrix. We then used the method to profile the dynamic interactome of lamin A/C in multiple cell and tissue types under various treatment conditions. The ability to detect proximal proteins and putative interactors in intact tissues, and to quantify changes caused by different conditions or in the presence of disease mutations, can provide a window into cell biology and disease pathogenesis.

A novel somatic mutation achieves partial rescue in a child with Hutchinson-Gilford progeria syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a fatal sporadic autosomal dominant premature ageing disease caused by single base mutations that optimise a cryptic splice site within exon 11 of the LMNA gene. The resultant disease-causing protein, progerin, acts as a dominant negative. Disease severity relies partly on progerin levels. METHODS AND RESULTS: We report a novel form of somatic mosaicism, where a child possessed two cell populations with different HGPS disease-producing mutations of the same nucleotide-one producing severe HGPS and one mild HGPS. The proband possessed an intermediate phenotype. The mosaicism was initially discovered when Sanger sequencing showed a c.1968+2T>A mutation in blood DNA and a c.1968+2T>C in DNA from cultured fibroblasts. Deep sequencing of DNA from the proband's blood revealed 4.7% c.1968+2T>C mutation, and 41.3% c.1968+2T>A mutation. CONCLUSIONS: We hypothesise that the germline mutation was c.1968+2T>A, but a rescue event occurred during early development, where the somatic mutation from A to C at 1968+2 provided a selective advantage. This type of mosaicism where a partial phenotypic rescue event results from a second but milder disease-causing mutation in the same nucleotide has not been previously characterised for any disease.

Cell size and fat content of dietary-restricted Caenorhabditis elegans are regulated by ATX-2, an mTOR repressor.

Dietary restriction (DR) is a metabolic intervention that extends the lifespan of multiple species, including yeast, flies, nematodes, rodents, and, arguably, rhesus monkeys and humans. Hallmarks of lifelong DR are reductions in body size, fecundity, and fat accumulation, as well as slower development. We have identified atx-2, the Caenorhabditis elegans homolog of the human ATXN2L and ATXN2 genes, as the regulator of these multiple DR phenotypes. Down-regulation of atx-2 increases the body size, cell size, and fat content of dietary-restricted animals and speeds animal development, whereas overexpression of atx-2 is sufficient to reduce the body size and brood size of wild-type animals. atx-2 regulates the mechanistic target of rapamycin (mTOR) pathway, downstream of AMP-activated protein kinase (AMPK) and upstream of ribosomal protein S6 kinase and mTOR complex 1 (TORC1), by its direct association with Rab GDP dissociation inhibitor ß, which likely regulates RHEB shuttling between GDP-bound and GTP-bound forms. Taken together, this work identifies a previously unknown mechanism regulating multiple aspects of DR, as well as unknown regulators of the mTOR pathway. They also extend our understanding of diet-dependent growth retardation, and offers a potential mechanism to treat obesity.

BAF-1 mobility is regulated by environmental stresses.

Barrier to autointegration factor (BAF) is an essential component of the nuclear lamina that binds lamins, LEM-domain proteins, histones, and DNA. Under normal conditions, BAF protein is highly mobile when assayed by fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We report that Caenorhabditis elegans BAF-1 mobility is regulated by caloric restriction, food deprivation, and heat shock. This was not a general response of chromatin-associated proteins, as food deprivation did not affect the mobility of heterochromatin protein HPL-1 or HPL-2. Heat shock also increased the level of BAF-1 Ser-4 phosphorylation. By using missense mutations that affect BAF-1 binding to different partners we find that, overall, the ability of BAF-1 mutants to be immobilized by heat shock in intestinal cells correlated with normal or increased affinity for emerin in vitro. These results show BAF-1 localization and mobility at the nuclear lamina are regulated by stress and unexpectedly reveal BAF-1 immobilization as a specific response to caloric restriction in C. elegans intestinal cells.

Lamins in development, tissue maintenance and stress.

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Reversal of age-dependent nuclear morphology by inhibition of prenylation does not affect lifespan in Caenorhabditis elegans.

Fibroblasts derived from Hutchinson-Gilford progeria syndrome (HGPS) patients and dermal cells derived from healthy old humans in culture display age-dependent progressive changes in nuclear architecture due to accumulation of farnesylated lamin A. Treating human HGPS cells or mice expressing farnesylated lamin A with farnesyl transferase inhibitors (FTIs) reverses nuclear phenotypes and extends lifespan. Aging adult Caenorhabditis elegans show changes in nuclear architecture resembling those seen in HGPS fibroblasts, as well as a decline in motility, phenotypes which are also inhibited by the FTI gliotoxin. However, it was not clear whether these effects were due to loss of farnesylation or to side effects of this drug. Here, we used a different FTI, manumycin or downregulated polyprenyl synthetase with RNAi to test the roles of farnesylation in C. elegans aging. Our results show that the age-dependent changes in nuclear morphology depend on farnesylation. We also demonstrate that inhibition of farnesylation does not affect motility or lifespan, suggesting that the effects of blocking protein prenylation on nuclear morphology could be separated from their effects on motility and lifespan. These results provide further understanding of the role of lamin and farnesylation in the normal aging process and in HGPS.

Gliotoxin reverses age-dependent nuclear morphology phenotypes, ameliorates motility, but fails to affect lifespan of adult Caenorhabditis elegans.

Specific mutations in human LMNA or loss of ZMPSTE26 activity cause abnormal processing of lamin A and early aging diseases, including Hutchinson Gilford progeria syndrome (HGPS). HGPS fibroblasts in culture undergo age-dependent progressive changes in nuclear architecture. Treating these cells with farnesyl transferase inhibitors (FTIs) reverse these nuclear phenotypes and also extend lifespan of mice HGPS models. Dermal cells derived from healthy old humans also accumulate the abnormally processed lamin A. However, the effect of FTIs on normal aging cells was not tested. Aging adult C. elegans cells show changes in nuclear architecture similar to HGPS fibroblasts and down regulating lamin expression in adult C. elegans reduces their lifespan. Here, we show that nuclei of adult C. elegans, in which lamin is down-regulated, have similar phenotypes to normal aging nuclei, but at an earlier age. We further show that treating adult C. elegans with the FTI gliotoxin reverses nuclear phenotypes and improves motility of aging worms. However, the average lifespan of the gliotoxin-treated animals was similar to that of untreated animals. These results suggest that lamins are involved in the process of normal aging in C. elegans.