Poly(GR) interacts with key stress granule factors promoting its assembly into cytoplasmic inclusions.

C9orf72 repeat expansions are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Poly(GR) proteins are toxic to neurons by forming cytoplasmic inclusions that sequester RNA-binding proteins including stress granule (SG) proteins. However, little is known of the factors governing poly(GR) inclusion formation. Here, we show that poly(GR) infiltrates a finely tuned network of protein-RNA interactions underpinning SG formation. It interacts with G3BP1, the key driver of SG assembly and a protein we found is critical for poly(GR) inclusion formation. Moreover, we discovered that N6-methyladenosine (m6A)-modified mRNAs and m6A-binding YTHDF proteins not only co-localize with poly(GR) inclusions in brains of c9FTD/ALS mouse models and patients with c9FTD, they promote poly(GR) inclusion formation via the incorporation of RNA into the inclusions. Our findings thus suggest that interrupting interactions between poly(GR) and G3BP1 or YTHDF1 proteins or decreasing poly(GR) altogether represent promising therapeutic strategies to combat c9FTD/ALS pathogenesis.

Proximity labeling reveals dynamic changes in the SQSTM1 protein network.

Sequestosome1 (SQSTM1) is an autophagy receptor that mediates degradation of intracellular cargo, including protein aggregates, through multiple protein interactions. These interactions form the SQSTM1 protein network that are mediated by SQSTM1 functional interaction domains, which include LIR, PB1, UBA and KIR. Despite various attempts to unravel the complexity of the SQSTM1 protein network, our understanding of the relationship of various components in cellular physiology and disease states continues to evolve. To investigate the SQSTM1 protein interaction network, we performed proximity profile labeling by fusing TurboID with the human protein SQSTM1 (TurboID::SQSTM1). This chimeric protein displayed well-established SQSTM1 features including: production of SQSTM1 intracellular bodies, binding to known SQSTM1 interacting partners via defined functional SQSTM1 interacting domains and capture of novel SQSTM1 interactors. Strikingly, aggregated tau protein altered the protein interaction network of SQSTM1 to include many stress-associated proteins. Overall, our work reveals the dynamic landscape of the SQSTM1 protein network and offers a resource to study SQSTM1 function in cellular physiology and disease state.

Protein Interaction Network Biology in Neuroscience.

Mapping the intricate networks of cellular proteins in the human brain has the potential to address unsolved questions in molecular neuroscience, including the molecular basis of cognition, synaptic plasticity, long-term potentiation, learning, and memory. Perturbations to the protein-protein interaction networks (PPIN) present in neurons, glia, and other cell-types have been linked to multifactorial neurological disorders. Yet while knowledge of brain PPINs is steadily improving, the complexity and dynamic nature of the heterogeneous central nervous system in normal and disease contexts poses a formidable experimental challenge. In this review, the recent applications of functional proteomics and systems biology approaches to study PPINs central to normal neuronal function, during neurodevelopment, and in neurodegenerative disorders are summarized. How systematic PPIN analysis offers a unique mechanistic framework to explore intra- and inter-cellular functional modules governing neuronal activity and brain function is also discussed. Finally, future technological advancements needed to address outstanding questions facing neuroscience are outlined.

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

Self-Assembly of an Organized Cementum-Periodontal Ligament-Like Complex Using Scaffold-Free Tissue Engineering.

A major challenge in regenerating periodontal tissues is emulating its complex structure containing both mineralized and soft tissues. In this study, scaffold-free tissue constructs engineered using periodontal ligament cells (PDLCs), which contain a population of adult stem/progenitor cells, self-assembled into an organized multi-tissue structure comprising a mineralized cementum-like core enclosed within a periodontal ligament (PDL)-like tissue. Scaffold-free engineered constructs were formed by culturing human PDLCs to form a cell sheet on six-well dishes containing two minutien pins placed 7 mm apart. The cell sheet was contracted by the cells to roll into the pins forming a cylindrical construct anchored on either end by the pins. These tissues were approximately 1 mm in diameter and 7 mm long and contained only the cells and their endogenous matrix. These scaffold-free engineered constructs exhibited two structurally distinct tissues, one in the center of the construct and another on the periphery. The center tissue was mineralized and expressed alkaline phosphatase and bone sialoprotein, similar to cementum. The peripheral tissue was not calcified and expressed periodontal ligament-associated protein-1 and periostin, which is characteristic of the periodontal ligament. This tissue organization was seen after in vitro culture and maintained in vivo following subcutaneous implantation in immunocompromised mice. These data demonstrate that scaffold-free tissue engineering facilitates PDLCs to self-assemble into an organized cementum-PDL-like complex. These engineered tissues could be used as implantable grafts to regenerate damaged periodontal tissues or as model systems to study PDLC biology and mechanisms driving organized tissue assembly within the periodontium.

Mechanical Properties and Composition of the Basal Leaflet-Annulus Region of the Tricuspid Valve.

The Tricuspid valve (TV) annulus is a transition structure from the leaflets to the myocardium, with 3 different annulus segments corresponding to the TV leaflets, which includes both basal leaflets and bordering myocardium. The objective of this study was to understand TV annulus mechanical properties and correlate it to the biological composition. The uniaxial testing of the annulus segments from ten porcine TVs was performed to measure Young's modulus (E) and extensibility (εT). Western blotting and histology were executed. The septal annulus E value (208.7 ± 67.2 kPa) was statistically greater (p < 0.01) than that of the anterior (92.0 ± 66.8 kPa) and the posterior annulus segment (136.8 ± 56.9 kPa) (p < 0.05), respectively. εT among the 3 segments were equivalent (p values < 0.05). Western blotting and histology indicated that collagen was greatest along the septal annulus segment, which is correlated to E values. Collagen fibers from the leaflets inserted into the myocardium and faded out. Collagen content explains greater E and suture strength in the surgical annulus repair and larger resistance to annulus dilation in the septal annulus as compared with other segments. This study elucidates new knowledge of mechanical properties of the basal leaflet-annulus region of the TV annulus, which can be useful for future TV repair techniques.

Annulus Tension on the Tricuspid Valve: An In-Vitro Study.

Annulus tension (AT) is defined as leaflet tension per unit length of the annulus circumference. AT was investigated to understand tricuspid valve (TV) annulus mechanics. Ten porcine TVs were mounted on a right ventricle rig with an annulus plate to simulate TV closure. The TVs were mounted on the annulus plate in a normal and dilated TV annulus sizes, and closed under transvalvular pressure of 40 mmHg with the annulus held peripherally by wires. Anterior papillary muscle (PM) and septal PM were displaced in the dilated annulus. Wire forces were measured, and ATs (N/m) were calculated. Clover repair was performed in the dilated TV state subsequently, and AT was calculated again. A one-way ANOVA and Tukey's HSD test were used to test significances between the different TV states along each annulus segment with p < 0.05. Average ATs for the normal annulus, dilated annulus, and clover repair were 10.75 ± 1.87, 28.81 ± 10.51, and 26.93 ± 11.44 N/m, respectively. Septal annulus segments had the highest ATs when compared to the other segments. For the clover repair, there were no significant changes in AT values. ATs and leaflet forces increased roughly 3-4 times with annulus dilation. AT decelerates annulus dilation as previously shown in the mitral valve. Clover repair does not prevent further annulus dilation by AT change and should be accompanied by annuloplasty. AT improves annulus contraction during a cardiac cycle and should be considered when designing annuloplasty in the future.

Identification of Response Options to Artisanal and Small-Scale Gold Mining (ASGM) in Ghana via the Delphi Process.

The Delphi technique is a means of facilitating discussion among experts in order to develop consensus, and can be used for policy formulation. This article describes a modified Delphi approach in which 27 multi-disciplinary academics and 22 stakeholders from Ghana and North America were polled about ways to address negative effects of small-scale gold mining (ASGM) in Ghana. In early 2014, the academics, working in disciplinary groups, synthesized 17 response options based on data aggregated during an Integrated Assessment of ASGM in Ghana. The researchers participated in two rounds of Delphi polling in March and April 2014, during which 17 options were condensed into 12. Response options were rated via a 4-point Likert scale in terms of benefit (economic, environmental, and benefit to people) and feasibility (economic, social/cultural, political, and implementation). The six highest-scoring options populated a third Delphi poll, which 22 stakeholders from diverse sectors completed in April 2015. The academics and stakeholders also prioritized the response options using ranking exercises. The technique successfully gauged expert opinion on ASGM, and helped identify potential responses, policies and solutions for the sector. This is timely given that improvement to the ASGM sector is an important component within the UN Minamata Convention.

Proteome analysis of the triton-insoluble erythrocyte membrane skeleton.

Erythrocyte shape and membrane integrity is imparted by the membrane skeleton, which can be isolated as a Triton X-100 insoluble structure that retains the biconcave shape of intact erythrocytes, indicating isolation of essentially intact membrane skeletons. These erythrocyte "Triton Skeletons" have been studied morphologically and biochemically, but unbiased proteome analysis of this substructure of the membrane has not been reported. In this study, different extraction buffers and in-depth proteome analyses were used to more fully define the protein composition of this functionally critical macromolecular complex. As expected, the major, well-characterized membrane skeleton proteins and their associated membrane anchors were recovered in good yield. But surprisingly, a substantial number of additional proteins that are not considered in erythrocyte membrane skeleton models were recovered in high yields, including myosin-9, lipid raft proteins (stomatin, flotillin1 and 2), multiple chaperone proteins (HSPs, protein disulfide isomerase and calnexin), and several other proteins. These results show that the membrane skeleton is substantially more complex than previous biochemical studies indicated, and it apparently has localized regions with unique protein compositions and functions. This comprehensive catalog of the membrane skeleton should lead to new insights into erythrocyte membrane biology and pathogenic mutations that perturb membrane stability. Biological significance Current models of erythrocyte membranes describe fairly simple homogenous structures that are incomplete. Proteome analysis of the erythrocyte membrane skeleton shows that it is quite complex and includes a substantial number of proteins whose roles and locations in the membrane are not well defined. Further elucidation of interactions involving these proteins and definition of microdomains in the membrane that contain these proteins should yield novel insights into how the membrane skeleton produces the normal biconcave erythrocyte shape and how it is perturbed in pathological conditions that destabilize the membrane.

Hemoglobin interacting proteins and implications of spectrin hemoglobin interaction.

In this report we have analyzed interacting partners of hemoglobin inside erythrocyte and sought possible implications of hemoglobin-spectrin interaction. Our list of identified cytosolic hemoglobin interacting proteins includes redox regulators like peroxiredoxin-2, Cu-Zn superoxide dismutase, catalase, aldehyde dehydrogenase-1, flavin reductase and chaperones like HSP70, α-hemoglobin stabilizing protein. Others include metabolic enzymes like carbonic anhydrase-1, selenium binding protein-1, purine nucleoside phosphorylase and nucleoside diphosphate kinase. Additionally, various membrane proteins like α and ß spectrin, ankyrin, band3, protein4.1, actin and glyceraldehyde 3 phosphate dehydrogenase have been shown to interact with hemoglobin. Our result indicates that major membrane skeleton protein spectrin, that also has a chaperone like activity, helps to fold the unstable alpha-globin chains in vitro. Taken together our results could provide insight into a protein network evolved around hemoglobin molecule inside erythrocyte that may add a new perspective in understanding the hemoglobin function and homeostasis.

2D DIGE based proteomics study of erythrocyte cytosol in sickle cell disease: altered proteostasis and oxidative stress.

Sickle cell disease (SCD) is a hemolytic disorder caused by a mutation in beta-globin gene and affects millions of people worldwide. Though clinical manifestations of the disease are quite heterogeneous, many of them occur due to erythrocyte sickling at reduced oxygen concentration and vascular occlusion mediated via blood cell adhesion to the vessel wall. We have followed proteomic approach to resolve the differentially regulated proteins of erythrocyte cytosol. The deregulated proteins mainly fall in the group of chaperone proteins such as heat shock protein 70, alpha hemoglobin stabilizing protein, and redox regulators such as aldehyde dehydrogenase and peroxiredoxin-2 proteoforms. Proteasomal subunits are found to be upregulated and phospho-catalase level also got altered. Severe oxidative stress inside erythrocyte is evident from the ROS analysis and Oxyblot(TM) experiments. Peroxiredoxin-2 shows significant dimerization in the SCD patients, a hallmark of oxidative stress inside erythrocytes. One interesting fact is that most of the differentially regulated proteins are also common for hemoglobinopathies such as Eß thalassemia. These could provide important clues in understanding the pathophysiology of SCD and lead us to better patient management in the future.

Elevated levels of redox regulators, membrane-bound globin chains, and cytoskeletal protein fragments in hereditary spherocytosis erythrocyte proteome.

OBJECTIVE: Hereditary spherocytosis (HS), a common inherited hemolytic anemia characterized by decreased deformability, reduced surface to volume ratio, and increased osmotic fragility of the spheroidal erythrocytes, is associated with several mutations of α- and ß-spectrin, ankyrin, band 3, band 4.2. HS manifests itself with high degrees of clinical heterogeneity and the molecular events leading to premature hemolysis of the spherocytes are unclear. We have employed proteomic techniques to identify differentially regulated proteins in the membrane and hemoglobin-depleted cytosol of HS erythrocytes. METHODS: We have employed 2-D gel electrophoresis and tandem matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry to investigate the differential proteome profiling of membrane and hemoglobin-depleted cytosol of erythrocytes isolated from the peripheral blood samples of HS patients and normal volunteers. RESULTS: Our study showed that redox regulators are up-regulated; while a co-chaperone and a nucleotide kinase are down-regulated in HS erythrocyte cytosol. We observed elevated levels of membrane-associated globin chains and low-molecular weight fragments of several major cytoskeletal proteins. CONCLUSION: The observed changes in the erythrocyte proteomes indicate altered redox regulation, nucleotide metabolism, protein aggregation and/or degradation, cytoskeletal disorganization, and severe oxidative stress in HS. Taken together, this study could enlighten upon disease progression and pathophysiology of HS.

vProtein: identifying optimal amino acid complements from plant-based foods.

BACKGROUND: Indispensible amino acids (IAAs) are used by the body in different proportions. Most animal-based foods provide these IAAs in roughly the needed proportions, but many plant-based foods provide different proportions of IAAs. To explore how these plant-based foods can be better used in human nutrition, we have created the computational tool vProtein to identify optimal food complements to satisfy human protein needs. METHODS: vProtein uses 1251 plant-based foods listed in the United States Department of Agriculture standard release 22 database to determine the quantity of each food or pair of foods required to satisfy human IAA needs as determined by the 2005 daily recommended intake. The quantity of food in a pair is found using a linear programming approach that minimizes total calories, total excess IAAs, or the total weight of the combination. RESULTS: For single foods, vProtein identifies foods with particularly balanced IAA patterns such as wheat germ, quinoa, and cauliflower. vProtein also identifies foods with particularly unbalanced IAA patterns such as macadamia nuts, degermed corn products, and wakame seaweed. Although less useful alone, some unbalanced foods provide unusually good complements, such as Brazil nuts to legumes. Interestingly, vProtein finds no statistically significant bias toward grain/legume pairings for protein complementation. These analyses suggest that pairings of plant-based foods should be based on the individual foods themselves instead of based on broader food group-food group pairings. Overall, the most efficient pairings include sweet corn/tomatoes, apple/coconut, and sweet corn/cherry. The top pairings also highlight the utility of less common protein sources such as the seaweeds laver and spirulina, pumpkin leaves, and lambsquarters. From a public health perspective, many of the food pairings represent novel, low cost food sources to combat malnutrition. Full analysis results are available online at http://www.foodwiki.com/vprotein.

Differential expression of red cell proteins in hemoglobinopathy.

Red blood cell proteome has not been studied well until recently, as the large abundance of hemoglobin posed challenge to the detection of other cytosolic proteins in the linear dynamic range. However, in the last couple of years, due to emergence of various novel hemoglobin depletion strategies and more state-of-the-art detection techniques, a number of works on erythrocyte proteome have appeared in the literature. As a result, we now have much deeper information about both the membrane as well as the cytosolic proteins of erythrocytes. In this review, we have discussed the role of red cell proteome on the two most well-studied hemoglobin disorders, sickle cell disease and thalassemia, emphasizing on the differential expression of the redox regulator proteins and chaperones, in particular. We have also touched upon the importance of the association of the varying levels of hemoglobin variants, particularly HbE on the clinical manifestation of composite diseases like HbEß thalassemia.