(DGIST (Daegu Gyeongbuk Institute of Science and Technology)) A drug-loaded microrobotic needle effectively targets and remains attached to cancerous tissue in lab experiments without needing continuous application of a magnetic field, allowing more precise drug delivery. The details were published by researchers at DGIST's Microrobot Research Center in Korea and colleagues in the journal Advanced Healthcare Materials.

(University of Jyväskylä - Jyväskylän yliopisto) Researchers at the universities of Jyvaskyla and Xiamen discovered a novel way to make functional macroscopic crystalline materials out of nanometer-size 34-atom silver-gold intermetallic clusters. The cluster material has a highly anisotropic electrical conductivity, being a semiconductor in one direction and an electrical insulator in other directions. The research was published in Nature Communications on May 6, 2020.

(Arizona State University) Cun-Zheng Ning, a professor of electrical engineering in the Ira A. Fulton Schools of Engineering at Arizona State University, and collaborators from Tsinghua University in China discovered a process of physics that enables low-power nanolasers to be produced in 2D semiconductor materials. Understanding the physics behind lasers at nanoscale and how they interact with semiconductors can have major implications for high-speed communication channels for supercomputers and data centers.

(Georgia Institute of Technology) The science behind sticky gecko's feet lets gecko adhesion materials pick up about anything. But cost-effective mass production of the materials was out of reach until now. A new method of making them could usher the spread of gecko-inspired grabbers to assembly lines and homes.

The Centre for Health Protection announced that as of 4pm today, no new COVID-19 cases have been recorded, leaving the number of confirmed cases in Hong Kong at 1,040 so far.

The centre again urged members of the public to maintain an appropriate social distance with other people in their daily lives to minimise the risk of infection.

People should go out less and avoid social activities such as having meals out or other gatherings to minimise the risk of coronavirus clusters emerging in the community.

As the COVID-19 situation remains severe and the number of cases reported around the world continues to rise, people are strongly urged to avoid all non-essential travel outside Hong Kong as well as maintain strict personal and environmental hygiene at all times.

Additionally, the Hospital Authority reported that there are currently 142 patients under isolation and that 920 patients have been discharged upon recovery.

For information and health advice on COVID-19, visit the Government's dedicated webpage.

(To watch the full press conference with sign language interpretation, click here.)

Chief Executive Carrie Lam today said because Hong Kong has not reported a local COVID-19 case for over two weeks and imported cases are low, some anti-epidemic measures can be lifted.

During a press conference, Mrs Lam outlined that unlike some European countries, Hong Kong did not need to go into lockdown to contain the spread of the disease.

“Hong Kong has never gone into a stage of a complete city lockdown. In some of the European countries where they practise a city lockdown, residents are simply not allowed to leave their homes, except for some very essential purposes. But we have never adopted that practice.

“And in fact, many renowned experts are now trying to study our situation - why does Hong Kong succeed in keeping the confirmed cases at a low level without drastic measures like a complete city lockdown. And I do think that is a very interesting topic for further research.”

Mrs Lam noted that the Government had adopted the “suppress and lift” strategy under which restrictions are implemented and lifted in accordance with the infection situation.

“The strategy that Hong Kong has been adopting - and advocated by some of our experts - is what we call a ‘suppress and lift strategy’.

“So in light of the number of confirmed cases and likelihood of the spread of the disease in the community, we will have to suppress in order to make sure that there will be no surge in the number of confirmed cases as we have seen in some neighbouring regions.

“When the situation of the infection stabilises, that is the time for lifting, that is, loosening a bit so that society can operate more normally, especially for the businesses and for individuals’ behaviour.”

The Chief Executive said the Government still needed to monitor the COVID-19 situation closely, even though it was in the stage of lifting restrictions.

“We are now right in the stage of lifting because we have not had a local case for 16 days already and the number of imported cases is very, very low.

“We are now quite confident that the system of testing and holding that we have put in place for all arrivals from overseas would enable us to control the number of imported cases. So this is a time for lifting and this afternoon we have announced a number of lifting measures.

“If the situation continues to stay at the current level - no local cases, very few imported cases - then at the end of the 14-day period, that is May 22, of course that would be the time for more relaxation.”

Mrs Lam added that if a local case suddenly surfaced, Hong Kong may have to go back to some suppression measures, which was why the Government had to monitor the situation closely so it could take the necessary and pertinent response measures.

(To watch the full press conference with sign language interpretation, click here.)

Chief Executive Carrie Lam today said that Hong Kong’s needy and disadvantaged will receive masks as part of the Government’s new mask distribution programme.

Mrs Lam made the announcement during a press conference this afternoon and explained that millions of donated masks will also be distributed to those in need.

“I have outlined six measures to distribute masks freely to the people of Hong Kong, and of course in so doing, we will take special account of the disadvantaged, the elderly and street sleepers. 

“So apart from being a member of the Hong Kong population, they will receive reusable masks. They will receive disposable masks. We have this mask distribution program together with a large number of non-governmental organisations, charity groups and self-help groups.

“So we will continue to work with them to distribute another three million masks, which were donated to us.”

Mrs Lam emphasised that should there be a shortage of masks set aside for the needy, the Government will use its own supply to cover the shortfall.

“I am announcing that if we run out of donated masks, but there is still a need from this disadvantaged groups, we will use the government masks - the masks that we procured which are supposed to be for our own use - and share these with the needy groups in society.

“That's a way to ensure that, in a public health situation that we are now in, the needs of the disadvantaged groups will be fully taken care of.”

In addition to distributing donated masks, the Government announced other measures on mask supplies that include handing out single-use and reusable masks to all Hong Kong residents and students in need.

Such measures also call for increasing the supply of masks to staff of elderly homes and cleaning workers employed by the Government's outsourced service contractors, as well as providing masks to private medical practitioners.

The Innovation & Technology Bureau announced that as of 3pm today, the CuMask online registration system had received over 500,000 registrations, covering close to 1.38 million registrants in total. 

The bureau also responded to reports concerning the purpose of information collection and security of the registration system.

It noted that information provided by citizens in obtaining the masks will not be used for other purposes and that the Government will ensure the retention period of the personal data is no longer than the time required for the purposes for which the data is used.

The bureau pointed out that the registration system for masks operates on the Government's private cloud to ensure the stability and security of the system. 

In order to prevent intrusion and data leakage, multiple security measures have been put in place in compliance with the Government Information Technology Security Policy & Guidelines. 

These measures include a firewall, intrusion detection, anti-bot technology and installation of the latest anti-virus software with regular updates of virus definitions. 

The service has also passed an information security risk assessment and audit before launch.

The Privacy Commissioner for Personal Data has been consulted on the system's personal data processing arrangements. 

The system has also passed an independent third-party privacy impact assessment to ensure that the relevant service and system comply with the Personal Data (Privacy) Ordinance.

The bureau further explained that citizens need to provide their Hong Kong identity card number and date of birth for the registration system to match data with the Immigration Department’s system.

The process will be used to ascertain whether the registrant is a Hong Kong resident and check against any duplicated registrations. 

The local mobile number serves as a way to receive SMS messages on registration results and delivery, while the name and local address of the main registrant serves to verify whether the address exists and for arranging delivery. 

The bureau emphasised that the purposes of information collection have been clearly displayed on the front of the registration page for citizens to browse before registration.

Meanwhile, the bureau clarified the online rumours regarding the manufacturer of CuMask, noting that the CuMask is not manufactured by the Sun Hing Knitting Factory Limited nor Nan Fung Group.

The procurement of raw materials, coordination of production, sterilisation and packaging of the CuMask are being handled by the Hong Kong Research Institute of Textiles & Apparel, it said.

The bureau expressed regret about the rumours.

The Centre for Health Protection announced that as of 4pm today, no new COVID-19 cases have been recorded, leaving the number of confirmed cases in Hong Kong at 1,040 so far.

The centre again urged members of the public to maintain an appropriate social distance with other people to minimise the risk of infection.

People should go out less and avoid social activities such as having meals out or other gatherings to minimise the risk of coronavirus clusters emerging in the community.

As the COVID-19 situation remains severe and the number of cases reported around the world continues to rise, people are strongly urged to avoid all non-essential travel outside Hong Kong as well as maintain strict personal and environmental hygiene at all times.

Additionally, the Hospital Authority reported that there are currently 127 patients under isolation and that 932 patients have been discharged upon recovery.

For information and health advice on COVID-19, visit the Government's dedicated webpage.

The CuMask online registration system received over 720,000 registrations, covering two million registrants in total on the first day of registration on May 6, the Innovation & Technology Bureau announced today.

The bureau said the response is overwhelming and it is encouraged to see support for local invention.

"Our thanks go to support from all sides, including the Hong Kong Research Institute of Textiles & Apparel (HKRITA) which has been commissioned to oversee the project, the Crystal International Group Limited which is responsible for the production, the Novetex Textiles Limited in Tai Po Industrial Estate for providing clean room for sterilisation, The Mills and the TAL Apparel Limited for lending premises to set up workshops as well as the frontline workmen for their hard work over the past few months.

“This unrivalled challenge cannot be met without their joint efforts and the collaboration of the industry and our team,” the bureau stated.

The bureau commissioned the HKRITA to oversee the CuMask project in order to meet the imminent needs for masks in Hong Kong.

It pointed out that the Government Stores & Procurement Regulations do allow direct purchase to be made under extreme urgency.

The whole procurement process was conducted in accordance with the Government's procurement regulations and procedures and with confirmation that the conditions under the Agreement on Government Procurement of the World Trade Organization could be met.

The bureau further explained that in February and March this year, the Government contacted various suppliers of reusable masks. However, most stated that they had either stopped production, did not have enough stock, were unable to export materials due to export control, or unable to produce testing certification.

The epidemic at that time was serious and the supplies of anti-epidemic items were becoming scarce. Hong Kong did not have any raw materials or production lines.

Taking into account the aggressive procurement actions of anti-epidemic items by different countries, export control and suspension of production lines all over the world, the Government had to consider urgently the feasibility of manufacturing reusable masks that would be up to standard for use by the whole community.

On reviewing the reusable mask developed by the HKRITA, the Government considered that the design of the mask and materials used could meet the requirement, as there were supporting certifications proving its compliance with relevant international standards.

As for mass production, it depends on the availability of supply of raw materials. Having wide network in the industry, the HKRITA was able to acquire quality raw materials within a short period and put production lines in place.

The Government therefore commissioned the HKRITA to oversee the coordination of production through direct purchase with a view to supplying reusable masks to all Hong Kong residents as soon as possible.

The bureau added the HKRITA is a non-profit-making R&D centre fully subsidised by the Government, with most of their R&D projects funded by the Innovation & Technology Fund.

The HKRITA oversees the CuMask project on a non-profit-making basis. All expenses will be reimbursed to the HKRITA on the basis of actual spending.

People concerned with the effectiveness of the CuMask may browse the website for testing reports and patent information.

The Centre for Health Protection today announced it is investigating four additional confirmed COVID-19 cases.

The newly reported cases involve four males aged between 11 and 47. All of them travelled during the incubation period.

Epidemiological investigations and relevant contact tracing on the confirmed cases are ongoing.

The centre again urged the public to maintain an appropriate social distance from other people as much as possible to minimise the risk of infection.

People should go out less and avoid social activities such as having meals out or other gatherings to minimise the risk of outbreak clusters emerging in the community.

As the COVID-19 situation remains severe and the number of cases reported around the world continues to rise, people are strongly advised to avoid all non-essential travel outside Hong Kong as well as maintain strict personal and environmental hygiene at all times.

Additionally, the Hospital Authority reported that there are currently 120 patients under isolation and that 944 patients have been discharged upon recovery.

For information and health advice on COVID-19, visit the Government's dedicated webpage.

(To watch the full press briefing with sign language interpretation, click here.)

The Centre for Health Protection today said recovered COVID-19 patients or those who did not have any symptoms may be discharged from hospital 10 days after the onset of symptoms or a positive test result.

Its Communicable Disease Branch Head Dr Chuang Shuk-kwan told a press briefing that the revised discharge criteria was based on the latest scientific evidence.

“Our Scientific Committee on Emerging & Zoonotic Diseases met yesterday and examined the latest scientific evidence on whether the virus will be viable from a patient.

“And the available evidence showed that this virus is usually not detected after 10 days since the onset of symptoms of patients. Some patients may have persistent positive PCR (polymerase chain reaction) for a long period of time.”

Dr Chuang noted that patients still had to meet the criteria of having two clinical specimens test negative, or testing positive for the SARS-CoV-2 antibody to be discharged.

“We have revised the discharge criteria to include the patient who (must have) been staying in the hospital for at least 10 days after the onset of symptoms. So this is the additional criteria, in addition to the previous criteria of two consecutive negative specimens.

“We added another criteria (which is) in case a patient has stayed in the hospital for a long time, more than 10 days since the onset of symptoms, but he or she has persistent positive PCR despite the Ct (cycle threshold) value being very high, they can check their serology, the antibody. So if the antibody turns positive, usually it is after 10 days, then he or she can be discharged.

“So this is based on the latest scientific evidence.”

Premises can still operate other licensed businesses which are not required to be suspended if they were operating more than one licensed business before the closure, the Food & Health Bureau said today.

The bureau made the statement in response to media enquiries on some anti-epidemic measures which were relaxed from today.

The statement noted that in accordance with the Prevention & Control of Disease (Requirements & Directions) (Business & Premises) Regulation, the Secretary for Food & Health has issued directions by notices in the Gazette that certain scheduled premises, namely karaoke establishments, clubs or nightclubs, party rooms and bathhouses, should remain closed until May 21.

These scheduled premises may still operate other licensed businesses if the operators have implemented all measures to effectively stop or avoid operation of businesses and offering services which are required to be suspended.

For example, premises originally operated as karaoke establishments and catering businesses can continue their catering business in accordance with the relevant directions if all karaoke operation and services are suspended.

The directions state that facilities, installations and equipment for karaoke activities are closed or properly sealed off and notices are posted in prominent locations at the entrances clearly indicating that only catering services but no karaoke services are provided in the premises.

Other scheduled premises operating more than one licensed business can adopt similar measures to operate other licensed businesses which are not required to be suspended, the bureau added.

The Centre for Health Protection announced that as of 4pm today, no new COVID-19 cases have been recorded, leaving the number of confirmed cases in Hong Kong at 1,044 so far.

The centre again urged members of the public to maintain an appropriate social distance with other people to minimise the risk of infection.

It said people should go out less and avoid social activities such as having meals out or other gatherings to minimise the risk of coronavirus clusters emerging in the community.

As the COVID-19 situation remains severe and the number of cases reported around the world continues to rise, people are strongly urged to avoid all non-essential travel outside Hong Kong as well as maintain strict personal and environmental hygiene.

Additionally, the Hospital Authority reported that there are currently 109 patients under isolation and that 960 patients have been discharged upon recovery.

For information and health advice on COVID-19, visit the Government's dedicated webpage.

Subsidies to local primary producers and wholesale traders operating in fresh food wholesale markets are being disbursed from today, the Agriculture, Fisheries & Conservation Department announced.

The department said 2,847 applications for the subsidy scheme of $10,000 to each local primary producer under the second round of the Anti-epidemic Fund have been received, with 1,294 approved involving a total of $12,940,000.

A total of 346 applications to the scheme to provide a subsidy of $40,000 to each eligible wholesale trader operating in fresh food wholesale markets were also received with 148 approved involving a total of $5,920,000.

The application periods of the two schemes will end on June 1.

Additionally, 57 borrowers have participated in the arrangement of a one-off interest-free deferral of loan repayment for one year under the Fisheries Development Loan Fund, it said.

More than 8,200 applications for the Anti-epidemic Support Scheme for Property Management Sector (ASPM) have been received, with over 3,850 approved, the Government announced today.

The approved applications involve subsidies of more than $140 million and will benefit around 22,000 building blocks and about 35,750 frontline property management workers.

Launched under the Anti-epidemic Fund, the ASPM provides subsidies to owners' organisations or property management companies of eligible buildings to provide hardship allowance to frontline property management workers.

It also provides the Anti-epidemic Cleansing Subsidy to owners' organisations or property management companies.

The scheme’s first phase covers private residential and composite buildings, while its second phase covers industrial and commercial buildings.

The ASPM is still open for applications and continues to disburse subsidies.

Contact the Property Management Services Authority at 3696 1156 or 3696 1166, or visit its website for details.

The Centre for Health Protection announced that as of 4pm today, no new COVID-19 cases have been recorded, leaving the number of confirmed cases in Hong Kong at 1,044 so far.

The centre again urged the public to maintain an appropriate social distance with other people to minimise the risk of infection.

It said people should go out less and avoid social activities such as having meals out or other gatherings to minimise the risk of coronavirus clusters emerging in the community.

As the COVID-19 situation remains severe and the number of cases reported around the world continues to rise, people are strongly urged to avoid all non-essential travel outside Hong Kong as well as maintain strict personal and environmental hygiene.

Additionally, the Hospital Authority reported that there are currently 90 patients under isolation and that 967 patients have been discharged upon recovery.

For information and health advice on COVID-19, visit the Government's dedicated webpage.

We've all been saying it: These are unprecedented times. The impacts of the COVID-19 pandemic are incredibly wide-ranging and affect all facets of life. One that is hitting the scientific community very hard is the cancellation of meetings, large and small. While we are well-versed in connecting with colleagues and collaborators across a variety of online platforms, these do not replace the immensely gratifying aspects of attending meetings in person: the pleasure of catching up with old friends and making new ones, the insights gained from having real-time conversations with others working on the same topic but with different expertise and perspectives, and the stimulating new scientific ideas we carry home. We have all been feeling the disappointment as we learn that one meeting after another is forced to cancel, from the vibrant ASBMB annual meeting to summer conferences of all types.Another loss from the appropriate but painful decision to cancel the ASBMB annual meeting was the chance to hear from our Herbert Tabor Early Career Investigator Awardees, who represent the best science published in JBC in the preceding year. This year, the competition was particularly fierce. We hope and anticipate that we will be able to hear from the winners at next year's ASBMB annual meeting. But in the meantime, we want to raise a toast to Wenchao Zhao, Yue Yang, Manisha Dagar, Febin Varghese, and Ayumi Nagashima-Kasahara as our 2020 winners. We've captured their award-winning 2019 papers (1–5) on the JBC website (6), and extended profiles of the...

Extracytoplasmic sugar decoration of glycopolymer components of the bacterial cell wall contributes to their structural diversity. Typically, the molecular mechanism that underpins such a decoration process involves a three-component glycosylation system (TGS) represented by an undecaprenyl-phosphate (Und-P) sugar-activating glycosyltransferase (Und-P GT), a flippase, and a polytopic glycosyltransferase (PolM GT) dedicated to attaching sugar residues to a specific glycopolymer. Here, using bioinformatic analyses, CRISPR-assisted recombineering, structural analysis of cell wall–associated polysaccharides (CWPS) through MALDI-TOF MS and methylation analysis, we report on three such systems in the bacterium Lactococcus lactis. On the basis of sequence similarities, we first identified three gene pairs, csdAB, csdCD, and csdEF, each encoding an Und-P GT and a PolM GT, as potential TGS component candidates. Our experimental results show that csdAB and csdCD are involved in Glc side-chain addition on the CWPS components rhamnan and polysaccharide pellicle (PSP), respectively, whereas csdEF plays a role in galactosylation of lipoteichoic acid (LTA). We also identified a potential flippase encoded in the L. lactis genome (llnz_02975, cflA) and confirmed that it participates in the glycosylation of the three cell wall glycopolymers rhamnan, PSP, and LTA, thus indicating that its function is shared by the three TGSs. Finally, we observed that glucosylation of both rhamnan and PSP can increase resistance to bacteriophage predation and that LTA galactosylation alters L. lactis resistance to bacteriocin.

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo. Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

Hypersecretion of glucagon from pancreatic α-cells strongly contributes to diabetic hyperglycemia. Moreover, failure of α-cells to increase glucagon secretion in response to falling blood glucose concentrations compromises the defense against hypoglycemia, a common complication in diabetes therapy. However, the mechanisms underlying glucose regulation of glucagon secretion are poorly understood and likely involve both α-cell–intrinsic and intraislet paracrine signaling. Among paracrine factors, glucose-stimulated release of the GABA metabolite γ-hydroxybutyric acid (GHB) from pancreatic β-cells might mediate glucose suppression of glucagon release via GHB receptors on α-cells. However, the direct effects of GHB on α-cell signaling and glucagon release have not been investigated. Here, we found that GHB (4–10 μm) lacked effects on the cytoplasmic concentrations of the secretion-regulating messengers Ca2+ and cAMP in mouse α-cells. Glucagon secretion from perifused mouse islets was also unaffected by GHB at both 1 and 7 mm glucose. The GHB receptor agonist 3-chloropropanoic acid and the antagonist NCS-382 had no effects on glucagon secretion and did not affect stimulation of secretion induced by a drop in glucose from 7 to 1 mm. Inhibition of endogenous GHB formation with the GABA transaminase inhibitor vigabatrin also failed to influence glucagon secretion at 1 mm glucose and did not prevent the suppressive effect of 7 mm glucose. In human islets, GHB tended to stimulate glucagon secretion at 1 mm glucose, an effect mimicked by 3-chloropropanoic acid. We conclude that GHB does not mediate the inhibitory effect of glucose on glucagon secretion.

β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan using a modified recombinant endo-β-1,6-glucanase having diminished glucan hydrolase activity. The purified β-1,6-glucanase derivative bound to the β-1,6-glucan pustulan with a KD of 16.4 nm. We validated the specificity of this β-1,6-glucan probe by demonstrating its ability to detect cell wall β-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long β-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro. We also used this assay to measure β-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for β-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter PvvhBA, which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of PvvhBA, whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the PvvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of PvvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid β-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid β-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell–mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I–β2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV–PA123–130. We determined the structure of the BF2*1501–PA123–130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123–130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123–130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV–PA123–130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123–130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens.

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro. β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography–tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.

Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and short hairpin RNA–based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel–dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea32+ and CuB1+, and suggests that a peroxide-bound state (Fea33+–O−–O−–CuB2+) rather than an O2-bound state (Fea32+–O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fea32+–O2, whereas Fea33+–O−–O−–CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fea34+ = O2−, not as Fea34+–OH−. The distance suggests an ∼800-cm−1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+–O−–O−–CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

The C-type lectin receptors (CLRs) form a family of pattern recognition receptors that recognize numerous pathogens, such as bacteria and fungi, and trigger innate immune responses. The extracellular carbohydrate-recognition domain (CRD) of CLRs forms a globular structure that can coordinate a Ca2+ ion, allowing receptor interactions with sugar-containing ligands. Although well-conserved, the CRD fold can also display differences that directly affect the specificity of the receptors for their ligands. Here, we report crystal structures at 1.8–2.3 Å resolutions of the CRD of murine dendritic cell-immunoactivating receptor (DCAR, or Clec4b1), the CLR that binds phosphoglycolipids such as acylated phosphatidyl-myo-inositol mannosides (AcPIMs) of mycobacteria. Using mutagenesis analysis, we identified critical residues, Ala136 and Gln198, on the surface surrounding the ligand-binding site of DCAR, as well as an atypical Ca2+-binding motif (Glu-Pro-Ser/EPS168–170). By chemically synthesizing a water-soluble ligand analog, inositol-monophosphate dimannose (IPM2), we confirmed the direct interaction of DCAR with the polar moiety of AcPIMs by biolayer interferometry and co-crystallization approaches. We also observed a hydrophobic groove extending from the ligand-binding site that is in a suitable position to interact with the lipid portion of whole AcPIMs. These results suggest that the hydroxyl group-binding ability and hydrophobic groove of DCAR mediate its specific binding to pathogen-derived phosphoglycolipids such as mycobacterial AcPIMs.

Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β-fold, followed by a secretin/TonB, short N-terminal subdomain at the C terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pulldown assays, isothermal titration calorimetry, and thermal denaturation CD spectroscopy, we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation.

The Mycobacterium tuberculosis virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear. In the present study, using liposome-leakage and cytotoxicity assays, LC-MS/MS–based proteomics, and CCF-4 FRET analysis, we obtained evidence that the Nα-acetylation of the Thr-2 residue on EsxA, a post-translational modification that is present in mycobacteria but absent in Escherichia coli, is required for the EsxA:B separation. Substitutions at Thr-2 that precluded Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamics simulations revealed that at low pH, the Nα-acetylated Thr-2 makes direct and frequent “bind-and-release” contacts with EsxB, which generates a force that pulls EsxB away from EsxA. In summary, our findings provide evidence that the Nα-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

Fatty acid transport protein 2 (FATP2) is highly expressed in the liver, small intestine, and kidney, where it functions in both the transport of exogenous long-chain fatty acids and the activation of very-long-chain fatty acids. Here, using a murine model, we investigated the phenotypic impacts of deleting FATP2, followed by a transcriptomic analysis using unbiased RNA-Seq to identify concomitant changes in the liver transcriptome. WT and FATP2-null (Fatp2−/−) mice (5 weeks) were maintained on a standard chow diet for 6 weeks. The Fatp2−/− mice had reduced weight gain, lowered serum triglyceride, and increased serum cholesterol levels and attenuated dietary fatty acid absorption. Transcriptomic analysis of the liver revealed 258 differentially expressed genes in male Fatp2−/− mice and a total of 91 in female Fatp2−/− mice. These genes mapped to the following gene ontology categories: fatty acid degradation, peroxisome biogenesis, fatty acid synthesis, and retinol and arachidonic acid metabolism. Targeted RT-quantitative PCR verified the altered expression of selected genes. Of note, most of the genes with increased expression were known to be regulated by peroxisome proliferator–activated receptor α (PPARα), suggesting that FATP2 activity is linked to a PPARα-specific proximal ligand. Targeted metabolomic experiments in the Fatp2−/− liver revealed increases of total C16:0, C16:1, and C18:1 fatty acids; increases in lipoxin A4 and prostaglandin J2; and a decrease in 20-hydroxyeicosatetraenoic acid. We conclude that the expression of FATP2 in the liver broadly affects the metabolic landscape through PPARα, indicating that FATP2 provides an important role in liver lipid metabolism through its transport or activation activities.

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein–cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin–γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens–epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone–client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone–client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin–client complexes could contribute to lens aging and presbyopia.

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress–inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion–activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5–ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5–ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5–ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B'/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge–swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge–swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal–regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T50 values of ∼30–40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17β-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to its thermostability.

pncRNA-D is an irradiation-induced 602-nt long noncoding RNA transcribed from the promoter region of the cyclin D1 (CCND1) gene. CCND1 expression is predicted to be inhibited through an interplay between pncRNA-D and RNA-binding protein TLS/FUS. Because the pncRNA-D–TLS interaction is essential for pncRNA-D–stimulated CCND1 inhibition, here we studied the possible role of RNA modification in this interaction in HeLa cells. We found that osmotic stress induces pncRNA-D by recruiting RNA polymerase II to its promoter. pncRNA-D was highly m6A-methylated in control cells, but osmotic stress reduced the methylation and also arginine methylation of TLS in the nucleus. Knockdown of the m6A modification enzyme methyltransferase-like 3 (METTL3) prolonged the half-life of pncRNA-D, and among the known m6A recognition proteins, YTH domain-containing 1 (YTHDC1) was responsible for binding m6A of pncRNA-D. Knockdown of METTL3 or YTHDC1 also enhanced the interaction of pncRNA-D with TLS, and results from RNA pulldown assays implicated YTHDC1 in the inhibitory effect on the TLS–pncRNA-D interaction. CRISPR/Cas9-mediated deletion of candidate m6A site decreased the m6A level in pncRNA-D and altered its interaction with the RNA-binding proteins. Of note, a reduction in the m6A modification arrested the cell cycle at the G0/G1 phase, and pncRNA-D knockdown partially reversed this arrest. Moreover, pncRNA-D induction in HeLa cells significantly suppressed cell growth. Collectively, these findings suggest that m6A modification of the long noncoding RNA pncRNA-D plays a role in the regulation of CCND1 gene expression and cell cycle progression.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10–13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein–coupled receptors to intracellular effector molecules. In Drosophila, the Gαq gene encodes several Gαq splice variants, with the Gαq1 isoform protein playing a major role in fly phototransduction. However, Gαq1 null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. Here, we isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, we found that mutations in the Gαq gene disrupt light responses and demonstrate that the Gαq3 isoform protein is responsible for the residual light response in Gαq1 null mutants. Moreover, we report that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation stable Rh1-arrestin 2 (Arr2) complexes. Our findings clarify essential roles of several different Gαq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Gαq3 functions in rhodopsin synthesis.

VOLUME 292 (2017) PAGES 3531–3540This article has been withdrawn by Shuofeng Hu, Xiaomin Ying, Xiangming Zhang, and Ya Wang. Baocheng Hu, Xiang Wang, Ping Wang, Jian Wang, and Hongyan Wang could not be reached. In Fig. 1C, the DAPI and merged images for the no IR control were switched. The DNA-PKcs and actin immunoblots on the left appear to have been spliced. In Fig. 4C, the DNA-PKcs immunoblot appears to have been spliced. In Fig. 4D, lanes 1 and 5; lanes 2, 6, and 8; and lanes 3 and 7 of the DNA-PKcs immunoblot are the same. In the p-DNA-PKcs immunoblot, lanes 1 and 8, lanes 2 and 6, and lanes 3 and 7 are the same. In the CRY2 immunoblot, lanes 5 and 7 are the same. In the CDC25A immunoblot, lanes 3 and 8 are the same. In the GSK3B immunoblot, lanes 1 and 5 and lanes 3 and 7 are the same. Also in the GSK3B immunoblot, the upper GSK3B bands in lanes 6 and 8 are the same. Lanes 4 and 8 of the cyclin D1 immunoblot are the same. In Fig. 5A, the CDC25A immunoblot appears to have been spliced. Also in Fig. 5A, lanes 2–4 and lanes 6–8 of the CDC25A immunoblot are the same. Lanes 4–6 and 7–9 of the actin immunoblot are the same. In Fig. 5C, lane 1 of the CDC25A immunoblot was reused in lane 5, and lanes 3 and 4 were reused in lanes 7 and 8. In the...

VOLUME 289 (2014) PAGES 30635–30644This article has been withdrawn by Guangnan Chen, Dongkyoo Park, Francis A. Cucinotta, David S. Yu, Xingming Deng, William S. Dynan, Paul W. Doetsch, and Ya Wang. Hongyan Wang, Xiang Wang, Xiangming Zhang, and Xiaobing Tang could not be reached. The last two lanes of the actin immunoblot in Fig. 1A were reused in the last two lanes of the actin immunoblot in Fig. 1C. In Fig. 2A, the γ-H2AX and the merge with DAPI images for no IR treatment do not match. In Fig. 3A, lanes 3 and 4 of the γ-H2AX immunoblot were reused in lanes 7 and 8, and lanes 5 and 6 of the H2A immunoblot were reused in lanes 7 and 8. In Fig. 3B, lanes 5 and 6 of the H2A immunoblot were reused in lanes 7 and 8. In Fig. 3C, lanes 5 and 6 of the γ-H2AX immunoblot were reused in lanes 7 and 8. Additionally, lanes 1 and 2 of the H2A immunoblot were reused in lanes 3 and 4. In Fig. 3D, lanes 1 and 2 of the Mre11 immunoblot from lysates were reused in lanes 4 and 5. In the γ-H2AX immunoblot, lane 3 was reused in lane 7, and lane 4 was reused in lanes 6 and 8. Also in the H2A immunoblot, lanes 1 and 2 were reused in lanes 3 and 4. In Fig. 4B, lanes 2 and 6 of the Mre11 immunoblot from Ogg1−/− cells are the same. In the Ape1...