Set4 is a chromatin-associated protein, promotes survival during oxidative stress, and regulates stress response genes in yeast.

The Set4 protein in the yeast Saccharomyces cerevisiae contains both a PHD finger and a SET domain, a common signature of chromatin-associated proteins, and shares sequence homology with the yeast protein Set3, the fly protein UpSET, and the human protein mixed-lineage leukemia 5 (MLL5). However, the biological role for Set4 and its potential function in chromatin regulation has not been well defined. Here, we analyzed yeast cell phenotypes associated with loss of Set4 or its overexpression, which revealed that Set4 protects against oxidative stress induced by hydrogen peroxide. Gene expression analysis indicated that Set4 promotes the activation of stress response genes in the presence of oxidative insults. Using ChIP analysis and other biochemical assays, we also found that Set4 interacts with chromatin and directly localizes to stress response genes upon oxidative stress. However, recombinant Set4 did not show detectable methyltransferase activity on histones. Our findings also suggest that Set4 abundance in the cell is balanced under normal and stress conditions to promote survival. Overall, these results suggest a model in which Set4 is a stress-responsive, chromatin-associated protein that activates gene expression programs required for cellular protection against oxidative stress. This work advances our understanding of mechanisms that protect cells during oxidative stress and further defines the role of the Set3-Set4 subfamily of SET domain-containing proteins in controlling gene expression in response to adverse environmental conditions.

Dissecting human monoclonal antibody responses from mRNA- and protein-based XBB.1.5 COVID-19 monovalent vaccines.

The emergence of highly contagious and immune-evasive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has required reformulation of coronavirus disease 2019 (COVID-19) vaccines to target those new variants specifically. While previous infections and booster vaccinations can enhance variant neutralization, it is unclear whether the monovalent version, administered using either mRNA or protein-based vaccine platforms, can elicit de novo B-cell responses specific for Omicron XBB.1.5 variants. Here, we dissected the genetic antibody repertoire of 603 individual plasmablasts derived from five individuals who received a monovalent XBB.1.5 vaccination either with mRNA (Moderna or Pfizer/BioNtech) or adjuvanted protein (Novavax). From these sequences, we expressed 100 human monoclonal antibodies and determined binding, affinity and protective potential against several SARS-CoV-2 variants, including JN.1. We then select two vaccine-induced XBB.1.5 mAbs, M2 and M39. M2 mAb was a de novo, antibody, i.e., specific for XBB.1.5 but not ancestral SARS-CoV-2. M39 bound and neutralized both XBB.1.5 and JN.1 strains. Our high-resolution cryo-electron microscopy (EM) structures of M2 and M39 in complex with the XBB.1.5 spike glycoprotein defined the epitopes engaged and revealed the molecular determinants for the mAbs' specificity. These data show, at the molecular level, that monovalent, variant-specific vaccines can elicit functional antibodies, and shed light on potential functional and genetic differences of mAbs induced by vaccinations with different vaccine platforms.\.

Epitope-directed anti-SARS-CoV-2 scFv engineered against the key spike protein region could block membrane fusion.

The newly emerged SARS-CoV-2 causing coronavirus disease (COVID-19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2' site, by the TMPRSS2 protease. S2' cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2' site to be conserved among all the SARS-CoV-2 variants, we chose an S2' epitope encompassing the S2' cleavage site and generated single-chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2' epitope was determined. Incidentally, S2' epitope in the scFv bound structure adopts an alpha-helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody-S2' epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2' cleavage competitively.

Dynamics of lipid displacement inside the hydrophobic cavity of a nonspecific lipid transfer protein from Solanum melongena.

Nonspecific lipid transfer proteins are multifunctional and multispecific seed proteins with a characteristic hydrophobic cavity that runs form N-terminal to the C-terminal end. They are capable of binding and transferring different lipid molecules by means of their hydrophobic cavity. Apart from the cavity, lipid molecules bind and interact at key positions on the nsLTP surface as well. The plasticity of the hydrophobic cavity is an unusual property, considered as the primary lipid binding site. Here, we report a crystal structure of nsLTP from Solanum melongena with two lauric acid molecules bound inside the cavity. It has been observed that the extent of the N-terminal entry point and plasticity of the cavity can be extended, upon binding of one or two lipid molecules inside the cavity. The MD simulation further revealed that the lipid molecule shows high mobility inside the cavity and interestingly, was able to change its orientation. An alternate lipid entry site adjacent to the N-terminal end was uncovered during simulation and Arg-84 was implicated to be a potential regulatory residue aside from Tyr-59. Collectively, this study helps to understand that changes in orientation of the lipid inside the cavity could occur intermittently besides entering the cavity via tail-in-mechanism.Communicated by Ramaswamy H. Sarma.

Antibody multispecificity: A necessary evil?

Antibodies represent key effectors of the adaptive immune system. The specificity of antibodies is an established hallmark of the immune response. However, a certain proportion of antibodies exhibit limited promiscuity or multireactivity. Germline antibodies display plasticity which imparts multispecificity to enhance the antibody repertoire. Surprisingly, even affinity matured antibodies display such plasticity and multireactivity enabling their binding to more than one antigen. We propose that antibody multispecificity is a physiological requirement to expand the antibody repertoire at the germline level and to tolerate plasticity in antigens at the mature level. This property of the humoral immune response may attenuate the ability of infectious RNA viruses such as influenza, HIV and SARS-CoV-2 to acquire mutations that render resistance to neutralizing antibodies.

Sensory Changes Related to Dental Implant Placement: A Scoping Review.

AIMS: To perform a scoping review of the literature to elucidate the occurrence of nerve damage related to dental implant placement and the factors causing the sensory changes. METHODS: An extensive electronic search was conducted using the Cochrane Library, Medline via Ovid, PubMed, Wiley Online, Science Direct, CINAHL, and the Google Scholar databases from the year 1950 to 2020. RESULTS: The search resulted in 1,067 articles, out of which 76 were selected for this review. The articles were categorized as literature review articles, retrospective studies, prospective studies, and case series/case reports. Altogether, 2,526 subjects were assessed retrospectively, with 5.27% transient and 1.39% persistent sensory changes, and a cohort of 2,750 subjects were followed prospectively, with 6.22% transient and 1.31% persistent sensory changes. A total of 336 subjects were enrolled in various case reports and case series, with 5.95% transient sensory changes and 84.52% persistent neurosensory changes. The articles included were not of high quality and have variations in their study designs and reporting procedures, with limited sensory change data to include in this study. CONCLUSION: After surgical placement of dental implants in 5,612 patients, the incidence of transient sensory changes was 5.63%, and the incidence of persistent sensory changes was 6.33%. Factors affecting the incidence were: mandibular location of the implant, with the inferior alveolar nerve as the most commonly affected nerve. The common symptoms reported were paresthesia and dysesthesia. Age and gender were among other factors, for which data were not available in all the articles.

Immunoaffinity purification of endogenous proteins from S. cerevisiae for post-translational modification and protein interaction analysis.

Protein regulation by post-translational modifications and protein-protein interactions is critical to controlling molecular pathways. Here, we describe an immunoaffinity purification approach in Saccharomyces cerevisiae. The protocol uses an endogenously-expressed epitope-tagged protein and can be applied to the identification of post-translational modifications or protein binding partners. The lysine methyltransferase Set5 is used as an example here to purify phosphorylated Set5 and identify phosphosites; however, this approach can be applied to a diverse set of proteins in yeast. For complete details on the use and execution of this protocol, please refer to Jaiswal et al. (2020).

Set4 regulates stress response genes and coordinates histone deacetylases within yeast subtelomeres.

The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status identified a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, where the loss of Set4 decreases cell fitness and cell wall integrity. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for Set4 in regulating repressive, heterochromatin-like environments.

Function of the MYND Domain and C-Terminal Region in Regulating the Subcellular Localization and Catalytic Activity of the SMYD Family Lysine Methyltransferase Set5.

SMYD lysine methyltransferases target histones and nonhistone proteins for methylation and are critical regulators of muscle development and implicated in neoplastic transformation. They are characterized by a split catalytic SET domain and an intervening MYND zinc finger domain, as well as an extended C-terminal domain. Saccharomyces cerevisiae contains two SMYD proteins, Set5 and Set6, which share structural elements with the mammalian SMYD enzymes. Set5 is a histone H4 lysine 5, 8, and 12 methyltransferase, implicated in the regulation of stress responses and genome stability. While the SMYD proteins have diverse roles in cells, there are many gaps in our understanding of how these enzymes are regulated. Here, we performed mutational analysis of Set5, combined with phosphoproteomics, to identify regulatory mechanisms for its enzymatic activity and subcellular localization. Our results indicate that the MYND domain promotes Set5 chromatin association in cells and is required for its role in repressing subtelomeric genes. Phosphoproteomics revealed extensive phosphorylation of Set5, and phosphomimetic mutations enhance Set5 catalytic activity but diminish its ability to interact with chromatin in cells. These studies uncover multiple regions within Set5 that regulate its localization and activity and highlight potential avenues for understanding mechanisms controlling the diverse roles of SMYD enzymes.

Azoospermic infertility is associated with altered expression of DNA repair genes.

Compelling evidence suggest that germs cells are predominantly sensitive to DNA damaging agents in comparison to other cells. High fidelity DNA repair in testicular cells thus becomes indispensable to preserve the genomic integrity for passing on to the progeny. Compromised DNA repair machinery in the testicular cells may result in impaired spermatogenesis and infertility. It remains unclear if the alterations in the expression of DNA repair genes correlate with azoospermia and male infertility. In the present study, 54 non-obstructive azoospermic infertile patients with hypospermatogenesis (HS, n = 26), maturation arrest (MA, n = 15), Sertoli cell only syndrome (SCOS, n = 13) and 14 controls with obstructive azoospermia, but normal spermatogenesis were recruited. Expression profiling of 84 DNA repair genes in testicular biopsy samples was performed using PCR array. Out of 84 genes, 27, 64 and 28 genes showed >5 fold down-regulation in the HS, MA and SCOS groups, respectively. On the basis of differential expression and their functional significance in spermatogenesis, ten genes (MSH2, BRIP1, CCNH, LIG4, MGMT, NTHL1, PMS1, DMC1, POLB and XPA) were selected for validation of transcript levels in a higher number of cases using RT-PCR, which corroborated the findings of array. Four genes (MSH2, LIG4, PMS1 and DMC1) were analyzed for protein levels using immunohistochemistry, which further validated the loss of DNA repair gene expression. Caspase-3 immunostaining showed that the loss of DNA repair correlated with increased testicular apoptosis in patients. Maturation arrest showed the highest apoptotic index with maximum number of downregulated genes. We conclude that the loss of DNA repair genes expression in testis correlates with increased apoptosis, azoospermia and infertility.

Choose Your Own Adventure: The Role of Histone Modifications in Yeast Cell Fate.

When yeast cells are challenged by a fluctuating environment, signaling networks activate differentiation programs that promote their individual or collective survival. These programs include the initiation of meiotic sporulation, the formation of filamentous growth structures, and the activation of programmed cell death pathways. The establishment and maintenance of these distinct cell fates are driven by massive gene expression programs that promote the necessary changes in morphology and physiology. While these genomic reprogramming events depend on a specialized network of transcription factors, a diverse set of chromatin regulators, including histone-modifying enzymes, chromatin remodelers, and histone variants, also play essential roles. Here, we review the broad functions of histone modifications in initiating cell fate transitions, with particular focus on their contribution to the control of expression of key genes required for the differentiation programs and chromatin reorganization that accompanies these cell fates.

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Repression of Middle Sporulation Genes in Saccharomyces cerevisiae by the Sum1-Rfm1-Hst1 Complex Is Maintained by Set1 and H3K4 Methylation.

The conserved yeast histone methyltransferase Set1 targets H3 lysine 4 (H3K4) for mono, di, and trimethylation and is linked to active transcription due to the euchromatic distribution of these methyl marks and the recruitment of Set1 during transcription. However, loss of Set1 results in increased expression of multiple classes of genes, including genes adjacent to telomeres and middle sporulation genes, which are repressed under normal growth conditions because they function in meiotic progression and spore formation. The mechanisms underlying Set1-mediated gene repression are varied, and still unclear in some cases, although repression has been linked to both direct and indirect action of Set1, associated with noncoding transcription, and is often dependent on the H3K4me2 mark. We show that Set1, and particularly the H3K4me2 mark, are implicated in repression of a subset of middle sporulation genes during vegetative growth. In the absence of Set1, there is loss of the DNA-binding transcriptional regulator Sum1 and the associated histone deacetylase Hst1 from chromatin in a locus-specific manner. This is linked to increased H4K5ac at these loci and aberrant middle gene expression. These data indicate that, in addition to DNA sequence, histone modification status also contributes to proper localization of Sum1 Our results also show that the role for Set1 in middle gene expression control diverges as cells receive signals to undergo meiosis. Overall, this work dissects an unexplored role for Set1 in gene-specific repression, and provides important insights into a new mechanism associated with the control of gene expression linked to meiotic differentiation.

Gr/gr deletions on Y-chromosome correlate with male infertility: an original study, meta-analyses, and trial sequential analyses.

We analyzed the AZFc region of the Y-chromosome for complete (b2/b4) and distinct partial deletions (gr/gr, b1/b3, b2/b3) in 822 infertile and 225 proven fertile men. We observed complete AZFc deletions in 0.97% and partial deletions in 6.20% of the cases. Among partial deletions, the frequency of gr/gr deletions was the highest (5.84%). The comparison of partial deletion data between cases and controls suggested a significant association of the gr/gr deletions with infertility (P = 0.0004); however, the other partial deletions did not correlate with infertility. In cohort analysis, men with gr/gr deletions had a relatively poor sperm count (54.20 ± 57.45 million/ml) in comparison to those without deletions (72.49 ± 60.06), though the difference was not statistically significant (p = 0.071). Meta-analysis also suggested that gr/gr deletions are significantly associated with male infertility risk (OR = 1.821, 95% CI = 1.39-2.37, p = 0.000). We also performed trial sequential analyses that strengthened the evidence for an overall significant association of gr/gr deletions with the risk of male infertility. Another meta-analysis suggested a significant association of the gr/gr deletions with low sperm count. In conclusion, the gr/gr deletions show a strong correlation with male infertility risk and low sperm count, particularly in the Caucasian populations.

Dysregulation of apoptotic pathway candidate genes and proteins in infertile azoospermia patients.

OBJECTIVE: To dissect the role of the apoptotic pathway and its regulation in the pathogenesis of male infertility in nonobstructive azoospermia. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Sixty-three infertile azoospermic patients with different histologic phenotypes were recruited (obstructive azoospermia, n = 16; hypospermatogenesis, n = 11; maturation arrest, n = 15; Sertoli cell only, n = 21). INTERVENTION(S): Testicular biopsies for histopathologic and expression analysis. MAIN OUTCOME MEASURE(S): Expression analysis by quantitative reverse transcription-polymerase chain reaction, protein localization by immunohistochemistry and apoptotic proteome array. RESULT(S): Results showed significantly increased expression of proapoptotic proteins like BAX, BAD, and BAK and comparatively lowered expression of antiapoptotic BCL2 and BCLW. Immunostaining revealed increased active caspase-3 activity and more TUNEL-positive cells in different impaired phenotypes as compared with normal. In addition, significantly increased m-RNA expression of TGFB1, P53, and FASLG along with significant down-regulation of VEGFA were observed. Expression of phosphorylated P53 at the S15 position and phosphorylated RAD17 at S635 was observed in cases with spermatogenic impairment at the translational level. CONCLUSION(S): The results clearly indicate increased levels of apoptosis along with its other regulatory factors. The balance between pro- (BAX and BAK) and antiapoptotic (BCL2 and BCLW) genes was disturbed, which may lead to altered apoptosis. Therefore, altered regulation of apoptosis might be associated with impaired spermatogenesis, eventually leading to male infertility.

Chromosome microarray analysis: a case report of infertile brothers with CATSPER gene deletion.

We present the case of two brothers who were referred to a male infertility clinic for infertility workup. Conventional chromosome analysis and Y chromosome microdeletions did not reveal any genetic alterations. We utilized the chromosome microarray analysis (CMA) to identify novel and common variations associated with this severely impaired spermatogenesis cases. CMA specific results showed a common deletion in the 15q15.3 region that harbors genes like CATSPER2, STRC and PPIP5K1 in both cases (M18 and M19). In addition we identified small duplication in X and 11 chromosomes of M19. This is the first familial case report from India on occurrence of CATSPER gene deletion in human male infertility.

One-carbon metabolism, spermatogenesis, and male infertility.

Balanced diet is the natural source of micronutrients, such as folate and vitamins, vital for proper functioning of the body. One-carbon metabolic pathway along with folate and other vitamins plays an important role in DNA synthesis and in the establishment of epigenetic modifications like DNA/histone methylation. Spermatogenesis involves distinct cellular, genetic, and chromatin changes during the course of production of male gamete sperm. Folate and normal activity of 1-carbon metabolic pathway enzymes are central to nucleotide synthesis, methylation, and maintenance of genomic integrity as well as protection from DNA damage. As a result, polymorphisms in 1-carbon metabolic pathway genes affecting several physiological processes also have an impact on spermatogenesis and may affect directly or indirectly quality of sperm. Alterations in these processes may be a consequence of additive effect resulting from altered expression of 1-carbon metabolic pathway genes and/or inadequate folate/micronutrients supplementation. The present review provides an overview of different cellular and molecular events regulated by 1-carbon metabolic pathway enzymes and their impact on male reproductive health. It also summarizes the different studies where polymorphisms in the enzymes of 1-carbon metabolic pathway or folate deficiency are associated with male infertility and future prospects.

Association of interleukin-1beta C + 3953T gene polymorphism with human male infertility.

Cytokines are involved in the regulation of spermatogenesis likely mediating the crosstalk among Sertoli and germ cells to facilitate germ cell movement across the seminiferous epithelium during cellular events such as germ cell differentiation. Members of the Interleukin-1 (IL-1) family are pleiotropic cytokines that are involved in inflammation, immunoregulation, and other homeostatic functions. Interleukin-1 alpha (IL-1α), IL-1ß, and the IL-1 antagonistic molecule (IL-1 Ra) are present in the testis under normal homeostasis and they further increase upon infection/inflammation. In the present study we have examined the association of C + 3953T polymorphism of the human IL-1B gene with human male infertility. The case control study comprised of two groups: 222 infertile patients and 230 fertile healthy control men. Genotyping for SNP C + 3953T IL-1B was carried out by polymerase chain reaction followed by analysis with specific endonucleases (PCR-RFLP). DNA sequencing was used to validate the PCR-RFLP results. The genotype frequencies of the IL-1B Taq C/T polymorphism were compared between infertile men and controls. The frequency was significantly higher in asthenozoospermic patients compared to fertile control men (odds ratio = 10.4, CI: 2.50- 43.96, p = 0.001). The C + 3953T of the IL-1B gene is associated with male infertility risk in the asthenozoospermic patients from an Indian population.

Association of the IL1RN gene VNTR polymorphism with human male infertility.

Interleukin-1 (IL-1) is a regulatory cytokine that plays an important role in the maintenance of the immune environment of the testis, regulation of junction dynamics and cell differentiation during spermatogenesis. Members of the IL-1 family are pleiotropic cytokines that are involved in inflammation, immunoregulation and other homeostatic functions in the body. IL-1α, IL-1ß, and the IL-1 receptor antagonistic molecule (IL-1 Ra) are expressed in the testis under normal homeostasis and they further increase upon infection/inflammation. In the present study we have examined the association of Variable Number Tandem Repeats (VNTR) polymorphism of the Interleukin-1 receptor antagonist gene (IL1RN) with human male infertility. The case-control study comprised of two groups: 331 idiopathic infertile patients and 358 fertile healthy men. The study indicates risk of IL1RN2 variant with male infertility (OR: 1.43, CI: 1.1546 to 1.7804, P = 0.001). To our best knowledge, this is the first report that links IL1RN VNTR polymorphism with human male infertility.