An inclusive approach to designing a multi-epitope chimeric vaccine for Taenia infections by integrating proteomics and reverse vaccinology

BackgroundSoil- and water-transmitted helminths are a major concern in the developing world due to their high prevalence. More than a quarter of the population were estimated to be infected with helminths in these endemic zones.Research designAn in silico approach was used to design a vaccine construct against the Taenia genus utilizing the proteomic information and evaluation of the construct using immune-informatics.ResultsOur study identified 451 conserved proteins in Taenia spp. using the existing proteome; out of these, 141 were found to be expressed in cysticerci. These proteins were screened for antigenic epitopes and a multi-subunit vaccine was constructed. The constructed vaccine was assessed for its efficacy in mounting the appropriate immune response. Our constructed vaccine showed stability and optimal performance against the TLR 4 receptor, which is reported to be upregulated in Taenia infections in hosts.ConclusionImmune-informatics tools help design vaccines for neglected diseases such as those attributed to helminths, which are known to cause widespread morbidity. Our vaccine construct holds tremendous potential in conferring protection against all Taenia spp. of clinical relevance to human

Autophagy Induction as a Therapeutic Strategy for Neurodegenerative Diseases.

Autophagy is a major, conserved cellular pathway by which cells deliver cytoplasmic contents to lysosomes for degradation. Genetic studies have revealed extensive links between autophagy and neurodegenerative disease, and disruptions to autophagy may contribute to pathology in some cases. Autophagy degrades many of the toxic, aggregate-prone proteins responsible for such diseases, including mutant huntingtin (mHTT), alpha-synuclein (α-syn), tau, and others, raising the possibility that autophagy upregulation may help to reduce levels of toxic protein species, and thereby alleviate disease. This review examines autophagy induction as a potential therapy in several neurodegenerative diseases-Alzheimer's disease, Parkinson's disease, polyglutamine diseases, and amyotrophic lateral sclerosis (ALS). Evidence in cells and in vivo demonstrates promising results in many disease models, in which autophagy upregulation is able to reduce the levels of toxic proteins, ameliorate signs of disease, and delay disease progression. However, the effective therapeutic use of autophagy induction requires detailed knowledge of how the disease affects the autophagy-lysosome pathway, as activating autophagy when the pathway cannot go to completion (e.g., when lysosomal degradation is impaired) may instead exacerbate disease in some cases. Investigating the interactions between autophagy and disease pathogenesis is thus a critical area for further research

Discovery of mechanisms regulating transcription factor EB (TFEB)

Transcription Factor EB (TFEB) is a key member of the MiT/TFE family of transcription factors that regulate the expression of genes involved in autophagy, lysosomal biogenesis, and metabolism. Dysregulation of TFEB activity and expression has been implicated in several pathologies, including lysosomal storage disorders, cancer, and neurodegenerative diseases. Therefore, precise regulation of TFEB activity is essential to maintain cellular homeostasis. Modulation of TFEB activity is a promising therapeutic strategy; however, further research is needed to determine the most effective approaches. In this study, we used a targeted proteomics approach to identify novel mechanisms of TFEB regulation. Specifically, we aimed to identify interacting partners of TFEB during amino acid starvation, a condition known to activate TFEB. Our analysis identified the deubiquitinating enzyme USP7 as one of the promising hits, which is known to play a critical role in regulating protein stability by removing ubiquitin molecules from target proteins destined for degradation. Despite some preliminary studies, our understanding of TFEB turnover remains limited. We therefore investigated the role of USP7 as a regulator of TFEB stability. Our results show that USP7 prevents proteasomal degradation of TFEB and thus regulates its turnover. Depletion or catalytic inhibition of USP7 leads to destabilisation of TFEB. Mechanistically, USP7 removes the K48-linked polyubiquitination chains from TFEB, a post-translational modification that has been characterised in this study. In addition, three lysine residues (K116, K264 and K274) were identified by mass spectrometry analysis as potential sites for TFEB ubiquitination. Mutation of these sites prevented TFEB ubiquitination and rendered it insensitive to the effects of USP7 inhibition, confirming USP7-mediated deubiquitination of TFEB at these sites and its subsequent stabilisation. Further investigation of the role of USP7 in TFEB signalling revealed that USP7 inhibition impaired autophagic flux and starvation-induced TFEB-mediated transcription of lysosomal genes. Interestingly, our preliminary data suggest that the interaction between TFEB and USP7 may depend on the phosphorylation status of TFEB, particularly at S122, an mTORC1-specific phosphorylation site. The discovery of USP7-mediated stabilisation of TFEB provides new insights into the regulation of TFEB and may allow more precise manipulation of its activity for therapeutic purposes. We also investigated O-GlcNAc transferase (OGT) as a potential regulator of TFEB based on the results of mass spectrometric analysis. Our results show that OGT modifies TFEB through a previously unknown post-translational modification for TFEB - O-GlcNAcylation. Our preliminary results suggest that OGT inhibition reduces the transcriptional activity of TFEB, and that OGT may play a role in stabilising TFEB protein levels

The Post-translational Regulation of Transcription Factor EB (TFEB) in Health and Disease

Transcription factor EB (TFEB) is a basic helix-loop-helix leucine-zipper transcription factor that acts as a master regulator of lysosomal biogenesis, lysosomal exocytosis, and macro-autophagy. TFEB contributes to a wide range of physiological functions, including mitochondrial biogenesis, and innate and adaptive immunity. As such, TFEB is an essential component of cellular adaptation to stressors, ranging from nutrient deprivation to pathogenic invasion. The activity of TFEB depends on its subcellular localisation, turnover, and DNA-binding capacity, all of which are regulated at the post-translational level. Pathological states are characterised by a specific set of stressors, which elicit post-translational modifications that promote gain or loss of TFEB function in the affected tissue. In turn, the resulting increase or decrease in survival of the tissue in which TFEB is more or less active, respectively, may either benefit or harm the organism as a whole. In this way, the post-translational modifications of TFEB account for its otherwise paradoxical protective and deleterious effects on organismal fitness in diseases ranging from neurodegeneration to cancer. In this review, we describe how the intracellular environment characteristic of different diseases alters the post-translational modification profile of TFEB, enabling cellular adaptation to a particular pathological state.We are grateful for funding from the UK Dementia Research Institute (funded by the MRC, Alzheimer’s Research UK and the Alzheimer’s Society), and the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). M.T. is funded by the Rosetrees Trust and the Cambridge University School of Clinical Medicine (James Baird Fund and F.E. Elmore Fund). S.K. is funded by the Cambridge Commonwealth, European & International Trust, the Nehru Trust for Cambridge University, and the Trinity-Henry Barlow Scholarship

The post‐translational regulation of transcription factor EB ( TFEB ) in health and disease

Funder: Rosetrees Trust (Rosetrees); doi: http://dx.doi.org/10.13039/501100000833Funder: UK Dementia Research Institute (UK DRI); doi: http://dx.doi.org/10.13039/501100017510Funder: MRC; doi: http://dx.doi.org/10.13039/100018645Funder: Alzheimer's Research UKFunder: Alzheimer's Society; doi: http://dx.doi.org/10.13039/501100000320Funder: Cambridge University School of Clinical MedicineFunder: James Baird FundFunder: F.E. Elmore FundFunder: Cambridge CommonwealthFunder: European & International TrustFunder: Nehru Trust for Cambridge UniversityFunder: Trinity‐Henry Barlow ScholarshipTranscription factor EB (TFEB) is a basic helix–loop–helix leucine zipper transcription factor that acts as a master regulator of lysosomal biogenesis, lysosomal exocytosis, and macro‐autophagy. TFEB contributes to a wide range of physiological functions, including mitochondrial biogenesis and innate and adaptive immunity. As such, TFEB is an essential component of cellular adaptation to stressors, ranging from nutrient deprivation to pathogenic invasion. The activity of TFEB depends on its subcellular localisation, turnover, and DNA‐binding capacity, all of which are regulated at the post‐translational level. Pathological states are characterised by a specific set of stressors, which elicit post‐translational modifications that promote gain or loss of TFEB function in the affected tissue. In turn, the resulting increase or decrease in survival of the tissue in which TFEB is more or less active, respectively, may either benefit or harm the organism as a whole. In this way, the post‐translational modifications of TFEB account for its otherwise paradoxical protective and deleterious effects on organismal fitness in diseases ranging from neurodegeneration to cancer. In this review, we describe how the intracellular environment characteristic of different diseases alters the post‐translational modification profile of TFEB, enabling cellular adaptation to a particular pathological state

Investigation of crystallinity, mechanical properties, fracture toughness and cell proliferation in plasma sprayed graphene nano platelets reinforced hydroxyapatite coating

Graphene nanoplatelets (GNPs) (0, 1 wt% and 2 wt%) reinforced hydroxyapatite (HA), denoted by HA, HA-1G and HA-2G respectively, coatings were fabricated on titanium substrate (Ti-6Al-4V) through atmospheric plasma spraying. The major parameters such as porosity, crystallinity, mechanical properties, toughness and cell proliferation were manipulated by varying plasma power from 15 kW to 35 kW and content of GNPs. For the coating synthesized at all plasma power, GNPs were found to be retained by Raman spectroscopy. GNPs reinforcement has led to an improvement in the crystallinity of the composite coatings as compared to HA coatings. On the contrary to it, increase in plasma power from 15 kW to 35 kW resulted in decrease in crystallinity for all three individual coating. Further, Increment in plasma power from 15 kW to 35 kW delivered a significant enhancement in hardness, elastic modulus and fracture toughness up to 81%, 149% and 282% respectively for HA-1 wt% GNPs coating, while it improved to 20%, 50% and 173% respectively on the addition of 2 wt% GNPs in HA coating fabricated at 35 kW. Enhancement in hardness, elastic modulus and fracture toughness was due to three simultaneous reasons: (1) Reduction in porosity (2) Uniform dispersion of GNPs and (3) Toughening mechanism offered by GNPs. Further, the addition of GNPs showed a remarkable improvement in the rate of cell proliferation in the HA coating. A detailed discussion over the reasons behind every results have been made profoundly

Human cytomegalovirus degrades DMXL1 to inhibit autophagy, lysosomal acidification, and viral assembly.

Human cytomegalovirus (HCMV) is an important human pathogen that regulates host immunity and hijacks host compartments, including lysosomes, to assemble virions. We combined a quantitative proteomic analysis of HCMV infection with a database of proteins involved in vacuolar acidification, revealing Dmx-like protein-1 (DMXL1) as the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants reveals the uncharacterized 7 kDa US33A protein as necessary and sufficient for DMXL1 degradation, which occurs via recruitment of the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). US33A-mediated DMXL1 degradation inhibits lysosome acidification and autophagic cargo degradation. Formation of the virion assembly compartment, which requires lysosomes, occurs significantly later with US33A-expressing virus infection, with reduced viral replication. These data thus identify a viral strategy for cellular remodeling, with the potential to employ US33A in therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease

Human cytomegalovirus degrades DMXL1 to inhibit autophagy, lysosomal acidification, and viral assembly

Human cytomegalovirus (HCMV) is an important human pathogen that regulates host immunity and hijacks host compartments, including lysosomes, to assemble virions. We combined a quantitative proteomic analysis of HCMV infection with a database of proteins involved in vacuolar acidification, revealing Dmx-like protein-1 (DMXL1) as the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants reveals the uncharacterized 7 kDa US33A protein as necessary and sufficient for DMXL1 degradation, which occurs via recruitment of the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). US33A-mediated DMXL1 degradation inhibits lysosome acidification and autophagic cargo degradation. Formation of the virion assembly compartment, which requires lysosomes, occurs significantly later with US33A-expressing virus infection, with reduced viral replication. These data thus identify a viral strategy for cellular remodeling, with the potential to employ US33A in therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease