Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII

Citation: Wei, W., Zheng, C. L., Zhu, M., Zhu, X. F., Yang, R. C., Misra, S., & Zhang, B. (2017). Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII. Scientific Reports, 7, 14. doi:10.1038/srep45033Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 3(10)-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 3(10)-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 3(10)-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A

Symmetry breaking during homodimeric assembly activates an E3 ubiquitin ligase

Citation: Ye, Z., Needham, P. G., Estabrooks, S. K., Whitaker, S. K., Garcia, B. L., Misra, S., . . . Camacho, C. J. (2017). Symmetry breaking during homodimeric assembly activates an E3 ubiquitin ligase. Scientific Reports, 7(1). doi:10.1038/s41598-017-01880-4C-terminus of Hsc/p70-Interacting Protein (CHIP) is a homodimeric E3 ubiquitin ligase.Each CHIP monomer consists of a tetratricopeptide-repeat (TPR), helix-turn-helix (HH), and U-box domain.In contrast to nearly all homodimeric proteins, CHIP is asymmetric.To uncover the origins of asymmetry, we performed molecular dynamics simulations of dimer assembly.We determined that a CHIP monomer is most stable when the HH domain has an extended helix that supports intra-monomer TPR-U-box interaction, blocking the E2-binding surface of the U-box.We also discovered that monomers first dimerize symmetrically through their HH domains, which then triggers U-box dimerization.This brings the extended helices into close proximity, including a repulsive stretch of positively charged residues.Unable to smoothly unwind, this conflict bends the helices until the helix of one protomer breaks to relieve the repulsion.The abrupt snapping of the helix forces the C-terminal residues of the other protomer to disrupt that protomer's TPR-U-box tight binding interface, swiftly exposing and activating one of the E2 binding sites.Mutagenesis and biochemical experiments confirm that C-terminal residues are necessary both to maintain CHIP stability and function.This novel mechanism indicates how a ubiquitin ligase maintains an inactive monomeric form that rapidly activates only after asymmetric assembly. © 2017 The Author(s)

A REVIEW OF MONKEYPOX DISEASE AND FUTURE TREATMENT OPTIONS

The Monkeypox virus (MPXV) causative agent for Monkeypox disease resembles a smallpox-like illness and can lead to a number of serious medical issues in humans. It is an enveloped double-stranded DNA virus and belongs to the Orthopoxvirus genus. Monkeypox cases have increased after the smallpox vaccine was no longer administered. Monkeypox did not really receive widespread attention until the 2003 US outbreak. The majority of monkeypox cases connected to the 2022 outbreak are being reported in nations surrounding Europe and in the western world. The neurological, respiratory, and gastrointestinal systems are all known to be impacted. There are currently no standardised or ideal guidelines for the clinical management of patients with monkeypox (MPX), especially in low-resource settings. Patient outcomes may also be poor and their illnesses may last a long time. The range of clinical manifestations, including complications and sequelae, as well as characteristics of the illness that may be indicators of illness severity and poor outcomes, must be better understood in order to improve care. Though more research is required before they can be used in an endemic setting, new therapeutics and vaccines offer hope for the treatment and prevention of monkeypox

Psoriasis-associated variant Act1 D10N with impaired regulation by Hsp90

Act1 is an essential adaptor molecule in IL-17-mediated signaling and is recruited to the IL-17 receptor upon IL-17 stimulation. Here, we report that Act1 is a client protein of the molecular chaperone, Hsp90. The Act1 variant (D10N) linked to psoriasis susceptibility is defective in its interaction with Hsp90, resulting in a global loss of Act1 function. Act1-/- mice modeled the mechanistic link between Act1 loss of function and psoriasis susceptibility. Although Act1 is necessary for IL-17-mediated inflammation, Act1-/- mice exhibited a hyper TH17 response and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17-signaling, IL-22 is the main contributor to skin inflammation, providing a molecular mechanism for the association of Act1 (D10N) with psoriasis susceptibility

Apical targeting of syntaxin 3 is essential for epithelial cell polarity

In polarized epithelial cells, syntaxin 3 localizes to the apical plasma membrane and is involved in membrane fusion of apical trafficking pathways. We show that syntaxin 3 contains a necessary and sufficient apical targeting signal centered around a conserved FMDE motif. Mutation of any of three critical residues within this motif leads to loss of specific apical targeting. Modeling based on the known structure of syntaxin 1 revealed that these residues are exposed on the surface of a three-helix bundle. Syntaxin 3 targeting does not require binding to Munc18b. Instead, syntaxin 3 recruits Munc18b to the plasma membrane. Expression of mislocalized mutant syntaxin 3 in Madin-Darby canine kidney cells leads to basolateral mistargeting of apical membrane proteins, disturbance of tight junction formation, and loss of ability to form an organized polarized epithelium. These results indicate that SNARE proteins contribute to the overall specificity of membrane trafficking in vivo, and that the polarity of syntaxin 3 is essential for epithelial cell polarization

Interactions between the quality control ubiquitin ligase CHIP and ubiquitin conjugating enzymes

<p>Abstract</p> <p>Background</p> <p>Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains.</p> <p>Results</p> <p>The 2.9 Å crystal structure of the CHIP U-box domain complexed with UbcH5a shows that CHIP binds to UbcH5 and Ubc13 through similar specificity determinants, including a key S-P-A motif on the E2 enzymes. The determinants make different relative contributions to the overall interactions between CHIP and the two E2 enzymes. CHIP undergoes auto-ubiquitination by UbcH5 but not by Ubc13-Uev1a. Instead, CHIP drives the formation of unanchored polyubiquitin by Ubc13-Uev1a. CHIP also interacts productively with the class III E2 enzyme Ube2e2, in which the UbcH5- and Ubc13-binding specificity determinants are highly conserved.</p> <p>Conclusion</p> <p>The CHIP:UbcH5a structure emphasizes the importance of specificity determinants located on the long loops and central helix of the CHIP U-box, and on the N-terminal helix and loops L4 and L7 of its cognate E2 enzymes. The S-P-A motif and other specificity determinants define the set of cognate E2 enzymes for CHIP, which likely includes several Class III E2 enzymes. CHIP's interactions with UbcH5, Ube2e2 and Ubc13-Uev1a are consistent with the notion that Ubc13-Uev1a may work sequentially with other E2 enzymes to carry out K63-linked polyubiquitination of CHIP substrates.</p

A swine arterivirus deubiquitinase stabilizes two major envelope proteins and promotes production of viral progeny.

Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell's exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly

Structural basis of nucleotide exchange and client binding by the Hsp70 cochaperone Bag2

Cochaperones are essential for Hsp70/Hsc70-mediated folding of proteins and include nucleotide exchange factors (NEF) that assist protein folding by accelerating ADP/ATP exchange on Hsp70. The cochaperone Bag2 binds misfolded Hsp70 clients and also acts as a NEF, but the molecular basis of its functions is unclear. We show that, rather than being a member of the Bag domain family, Bag2 contains a new type of Hsp70 NEF domain, which we call the “Brand New Bag” (BNB) domain. Free and Hsc70-bound crystal structures of Bag2-BNB show its dimeric structure in which a flanking linker helix and loop bind to Hsc70 to promote nucleotide exchange. NMR analysis demonstrates that the client-binding sites and Hsc70 interaction sites of Bag2-BNB overlap, and that Hsc70 can displace clients from Bag2-BNB, indicating a distinct mechanism for the regulation of Hsp-70-mediated protein folding by Bag2

Global estimates on the number of people blind or visually impaired by cataract: a meta-analysis from 2000 to 2020

Background: To estimate global and regional trends from 2000 to 2020 of the number of persons visually impaired by cataract and their proportion of the total number of vision-impaired individuals. Methods: A systematic review and meta-analysis of published population studies and gray literature from 2000 to 2020 was carried out to estimate global and regional trends. We developed prevalence estimates based on modeled distance visual impairment and blindness due to cataract, producing location-, year-, age-, and sex-specific estimates of moderate to severe vision impairment (MSVI presenting visual acuity &lt;6/18, ≥3/60) and blindness (presenting visual acuity &lt;3/60). Estimates are age-standardized using the GBD standard population. Results: In 2020, among overall (all ages) 43.3 million blind and 295 million with MSVI, 17.0 million (39.6%) people were blind and 83.5 million (28.3%) had MSVI due to cataract blind 60% female, MSVI 59% female. From 1990 to 2020, the count of persons blind (MSVI) due to cataract increased by 29.7%(93.1%) whereas the age-standardized global prevalence of cataract-related blindness improved by −27.5% and MSVI increased by 7.2%. The contribution of cataract to the age-standardized prevalence of blindness exceeded the global figure only in South Asia (62.9%) and Southeast Asia and Oceania (47.9%). Conclusions: The number of people blind and with MSVI due to cataract has risen over the past 30 years, despite a decrease in the age-standardized prevalence of cataract. This indicates that cataract treatment programs have been beneficial, but population growth and aging have outpaced their impact. Growing numbers of cataract blind indicate that more, better-directed, resources are needed to increase global capacity for cataract surgery.</p

Global age-sex-specific mortality, life expectancy, and population estimates in 204 countries and territories and 811 subnational locations, 1950–2021, and the impact of the COVID-19 pandemic: a comprehensive demographic analysis for the Global Burden of Disease Study 2021

Background: Estimates of demographic metrics are crucial to assess levels and trends of population health outcomes. The profound impact of the COVID-19 pandemic on populations worldwide has underscored the need for timely estimates to understand this unprecedented event within the context of long-term population health trends. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 provides new demographic estimates for 204 countries and territories and 811 additional subnational locations from 1950 to 2021, with a particular emphasis on changes in mortality and life expectancy that occurred during the 2020–21 COVID-19 pandemic period. Methods: 22 223 data sources from vital registration, sample registration, surveys, censuses, and other sources were used to estimate mortality, with a subset of these sources used exclusively to estimate excess mortality due to the COVID-19 pandemic. 2026 data sources were used for population estimation. Additional sources were used to estimate migration; the effects of the HIV epidemic; and demographic discontinuities due to conflicts, famines, natural disasters, and pandemics, which are used as inputs for estimating mortality and population. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate under-5 mortality rates, which synthesised 30 763 location-years of vital registration and sample registration data, 1365 surveys and censuses, and 80 other sources. ST-GPR was also used to estimate adult mortality (between ages 15 and 59 years) based on information from 31 642 location-years of vital registration and sample registration data, 355 surveys and censuses, and 24 other sources. Estimates of child and adult mortality rates were then used to generate life tables with a relational model life table system. For countries with large HIV epidemics, life tables were adjusted using independent estimates of HIV-specific mortality generated via an epidemiological analysis of HIV prevalence surveys, antenatal clinic serosurveillance, and other data sources. Excess mortality due to the COVID-19 pandemic in 2020 and 2021 was determined by subtracting observed all-cause mortality (adjusted for late registration and mortality anomalies) from the mortality expected in the absence of the pandemic. Expected mortality was calculated based on historical trends using an ensemble of models. In location-years where all-cause mortality data were unavailable, we estimated excess mortality rates using a regression model with covariates pertaining to the pandemic. Population size was computed using a Bayesian hierarchical cohort component model. Life expectancy was calculated using age-specific mortality rates and standard demographic methods. Uncertainty intervals (UIs) were calculated for every metric using the 25th and 975th ordered values from a 1000-draw posterior distribution. Findings: Global all-cause mortality followed two distinct patterns over the study period: age-standardised mortality rates declined between 1950 and 2019 (a 62·8% [95% UI 60·5–65·1] decline), and increased during the COVID-19 pandemic period (2020–21; 5·1% [0·9–9·6] increase). In contrast with the overall reverse in mortality trends during the pandemic period, child mortality continued to decline, with 4·66 million (3·98–5·50) global deaths in children younger than 5 years in 2021 compared with 5·21 million (4·50–6·01) in 2019. An estimated 131 million (126–137) people died globally from all causes in 2020 and 2021 combined, of which 15·9 million (14·7–17·2) were due to the COVID-19 pandemic (measured by excess mortality, which includes deaths directly due to SARS-CoV-2 infection and those indirectly due to other social, economic, or behavioural changes associated with the pandemic). Excess mortality rates exceeded 150 deaths per 100 000 population during at least one year of the pandemic in 80 countries and territories, whereas 20 nations had a negative excess mortality rate in 2020 or 2021, indicating that all-cause mortality in these countries was lower during the pandemic than expected based on historical trends. Between 1950 and 2021, global life expectancy at birth increased by 22·7 years (20·8–24·8), from 49·0 years (46·7–51·3) to 71·7 years (70·9–72·5). Global life expectancy at birth declined by 1·6 years (1·0–2·2) between 2019 and 2021, reversing historical trends. An increase in life expectancy was only observed in 32 (15·7%) of 204 countries and territories between 2019 and 2021. The global population reached 7·89 billion (7·67–8·13) people in 2021, by which time 56 of 204 countries and territories had peaked and subsequently populations have declined. The largest proportion of population growth between 2020 and 2021 was in sub-Saharan Africa (39·5% [28·4–52·7]) and south Asia (26·3% [9·0–44·7]). From 2000 to 2021, the ratio of the population aged 65 years and older to the population aged younger than 15 years increased in 188 (92·2%) of 204 nations. Interpretation: Global adult mortality rates markedly increased during the COVID-19 pandemic in 2020 and 2021, reversing past decreasing trends, while child mortality rates continued to decline, albeit more slowly than in earlier years. Although COVID-19 had a substantial impact on many demographic indicators during the first 2 years of the pandemic, overall global health progress over the 72 years evaluated has been profound, with considerable improvements in mortality and life expectancy. Additionally, we observed a deceleration of global population growth since 2017, despite steady or increasing growth in lower-income countries, combined with a continued global shift of population age structures towards older ages. These demographic changes will likely present future challenges to health systems, economies, and societies. The comprehensive demographic estimates reported here will enable researchers, policy makers, health practitioners, and other key stakeholders to better understand and address the profound changes that have occurred in the global health landscape following the first 2 years of the COVID-19 pandemic, and longer-term trends beyond the pandemic