The ubiquitous presence of SPFH (Stomatin, Prohibitin, Flotillin, HflK/HflC) proteins in all domains of life suggests that their function would be conserved. However, SPFH functions are diverse with ...organism-specific attributes. SPFH proteins play critical roles in physiological processes such as mechanosensation and respiration. Here, we characterize the stomatin ORF19.7296/SLP3 in the opportunistic human pathogen Candida albicans. Consistent with the localization of stomatin proteins, a Slp3p-Yfp fusion protein formed visible puncta along the plasma membrane. We also visualized Slp3p within the vacuolar lumen. Slp3p primary sequence analyses identified four putative S-palmitoylation sites, which may facilitate membrane localization and are conserved features of stomatins. Plasma membrane insertion sequences are present in mammalian and nematode SPFH proteins, but are absent in Slp3p. Strikingly, Slp3p was present in yeast cells, but was absent in hyphal cells, thus categorizing it as a yeast-phase specific protein. Slp3p membrane fluorescence significantly increased in response to cellular stress caused by plasma membrane, cell wall, oxidative, or osmotic perturbants, implicating SLP3 as a general stress-response gene. A slp3Δ/Δ homozygous null mutant had no detected phenotype when slp3Δ/Δ mutants were grown in the presence of a variety of stress agents. Also, we did not observe a defect in ion accumulation, filamentation, endocytosis, vacuolar structure and function, cell wall structure, or cytoskeletal structure. However, SLP3 over-expression triggered apoptotic-like death following prolonged exposure to oxidative stress or when cells were induced to form hyphae. Our findings reveal the cellular localization of Slp3p, and for the first time associate Slp3p function with the oxidative stress response.
Candida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate ...in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation.
Biofilm formation by the opportunistic fungal pathogen C. albicans is a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by C. albicans biofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during C. albicans biofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from C. albicans and also from additional pathogenic Candida species. Furthermore, SAP5 and SAP6 deletions confirm that both proteases are required for proper biofilm development in vitro and in vivo We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of Candida biofilm-associated infections.
Differential expression of extracellular proteases and endogenous protease inhibitors has been associated with distinct molecular subtypes of breast cancer. However, due to the tight ...post-translational regulation of protease activity, protease expression-level data alone are not sufficient to understand the role of proteases in malignant transformation. Therefore, we hypothesized that global profiles of extracellular protease activity could more completely reflect differences observed at the transcriptional level in breast cancer and that subtype-associated protease activity may be leveraged to identify specific proteases that play a functional role in cancer signaling. Here, we used a global peptide library-based approach to profile the activities of proteases within distinct breast cancer subtypes. Analysis of 3651 total peptide cleavages from a panel of well-characterized breast cancer cell lines demonstrated differences in proteolytic signatures between cell lines. Cell line clustering based on protease cleavages within the peptide library expanded upon the expected classification derived from transcriptional profiling. An isogenic cell line model developed to further interrogate proteolysis in the HER2 subtype revealed a proteolytic signature consistent with activation of TGF-β signaling. Specifically, we determined that a metalloprotease involved in TGF-β signaling, BMP1, was upregulated at both the protein (2-fold, P = 0.001) and activity (P = 0.0599) levels. Inhibition of BMP1 and HER2 suppressed invasion of HER2-expressing cells by 35% (P < 0.0001), compared to 15% (P = 0.0086) observed in cells where only HER2 was inhibited. In summary, through global identification of extracellular proteolysis in breast cancer cell lines, we demonstrate subtype-specific differences in protease activity and elucidate proteolysis associated with HER2-mediated signaling.
The ability to efficiently isolate antigen-specific B cells in high throughput will greatly accelerate the discovery of therapeutic monoclonal antibodies (mAbs) and catalyze rational vaccine ...development. Traditional mAb discovery is a costly and labor-intensive process, although recent advances in single-cell genomics using emulsion microfluidics allow simultaneous processing of thousands of individual cells. Here we present a streamlined method for isolation and analysis of large numbers of antigen-specific B cells, including next generation antigen barcoding and an integrated computational framework for B cell multi-omics. We demonstrate the power of this approach by recovering thousands of antigen-specific mAbs, including the efficient isolation of extremely rare precursors of VRC01-class and IOMA-class broadly neutralizing HIV mAbs.
Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to ...single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.
Misregulated proteolysis has been linked to the initiation and progression of a large number of pathological processes. Transcriptional analysis of primary tumors and cell lines in breast cancer ...identified differential expression of proteases and protease inhibitors associated with different molecular subtypes. However, due to the tight regulation of protease activity post-translationally, it is not sufficient to know protease expression levels alone. Here, we used an unbiased global protease profiling approach to analyze changes in extracellular proteolysis in an isogenic cell line model, MCF10A HER2, representative of the HER2 subtype, with the goal of identifying proteases contributing to the oncogenic potential of HER2 in breast cancer. HER2 expression revealed changes consistent with activation of TGF-β signaling. Specifically, we determined that a metalloprotease involved in TGF-β activation, BMP1, was upregulated at both the protein and activity level. In summary, through global identification of extracellular proteolysis, we elucidate proteolysis associated with HER2-mediated signaling.
Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically ...dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.
Hablar de educación implica hablar del futuro desde la perspectiva presente con la que contamos. Toda la información surge de las características de la educación actual, de las necesidades actuales ...de los seres humanos y cómo paulatinamente cambian, para obligarnos sutilmente a generar procesos de adaptación.
Dicha adaptación ocurre incluyendo los usuarios del sistema, que es la misma sociedad, y el sistema organizativo de la misma. La cultura organizacional, la gestión administrativa y las funciones sustantivas, entran todos a hacer parte de esos cambios que se requieren y que son postulados en el presente artículo.
Así, en este artículo y como producto de la reflexión, se plantea la idea de que las empresas, y en este caso, Instituciones de Educación Superior, requieren contar con vínculos ajustables operativos (VAO) en su diseño organizacional que le permita responder a las necesidades de la sociedad actual y de los jóvenes y niños de y el mundo que queremos ver en 50 años
Summary The prognosis of advanced pancreatic adenocarcinoma is still poor nowadays. Gemcitabine in monotherapy (30-min infusion) has been the standard of treatment during the last decade, and many ...clinical trials have failed to demonstrate an improvement in overall survival (OS) with the addition of different drugs to gemcitabine, including cetuximab and bevacizumab. Nevertheless, some modest but interesting advances have been provided by combinations such as gemcitabine–erlotinib, gemcitabine–capecitabine and gemcitabine plus a platinum salt. In spite of this, survival results remain disappointing. Further research focused on new combinations, incorporating the new targeted therapies and identifying potential predictive factors of response are required to be able to offer effective tailored therapies to these patients.
Polychlorinated biphenyls and organochlorine pesticides are persistent organic pollutants (POPs) that had been banned or restricted in many countries, including Spain. However, their ubiquity still ...poses environmental and human health threats.
To longitudinally explore public healthcare costs associated with long-term exposure to a mixture of 8 POPs in a cohort of residents of two areas of Granada Province, Southern Spain.
Longitudinal study in a subsample (n = 385) of GraMo adult cohort. Exposure assessment was performed by analyzing adipose tissue POP concentrations at recruitment. Average primary care (APC) and average hospital care (AHC) expenditures of each participant over 14 years were estimated using the data from their medical records. Data analyses were performed by robust MM regression, weighted quantile sum regression (WQS) and G-computation analysis.
In the adjusted robust MM models for APC, most POPs showed positive beta coefficients, being Hexachlorobenzene (HCB) significantly associated (β: 1.87; 95% Confidence interval (95%CI): 0.17, 3.57). The magnitude of this association increased (β: 3.72; 95%CI: 0.80, 6.64) when the analyses were restricted to semi-rural residents, where β-HCH was also marginally-significantly associated to APC (β: 3.40; 95%CI: −0.10, 6.90). WQS revealed a positive but non-significant mixture association with APC (β: 0.14; 95%CI: −0.06, 0.34), mainly accounted for by β-HCH (54%) and HCB (43%), that was borderline-significant in the semi-rural residents (β: 0.23; 95%CI: −0.01, 0.48). No significant results were observed in G-Computation analyses.
Long-term exposure to POP mixtures might represent a modifiable factor increasing healthcare costs, thus affecting the efficiency of the healthcare systems. However, and owing the complexity of the potential causal pathways and the limitations of the present study, further research is warranted to fully elucidate ascertain whether interventions to reduce human exposure should be considered in healthcare policies.
•Long-term exposure to POPs was estimated by analyzing 385 adipose tissue samples.•HCB was positively associated with primary healthcare costs.•β-HCH was also associated with primary healthcare costs in semi-rural residents.•HCB and β-HCH also explained most of the mixture effect observed.•POP exposure might represent a modifiable factor increasing healthcare costs.
PDF
