Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc),O-linked-GlcNAc transferase (OGT) catalyzes Ser/ThrO-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase-protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase-protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT

Region II CEE <i>O</i>-GlcNAcylation and HCF-1_PRO-repeat proteolysis are independent OGT activities.

<p>(A) (Left) The full-length (FL) HCF-1rep1 precursor (band a) and the N-terminal cleavage product (band b) were purified from HEK 293 lysates via α-HA-epitope immunoprecipitation and visualized by Coomassie staining. The bands were analyzed for <i>O</i>-GlcNAcylation and phosphorylation sites by LC-MS/MS. (Right) Schematic representation of identified HCF-1rep1 <i>O</i>-GlcNAcylation (squares) and phosphorylation (yellow circles) sites in the uncleaved HCF-1rep1 precursor. The HCF-1 sequences covered by the analysis (residues 867–1071) and the engineered trypsin cleavage sites A933K and M951K are indicated below the diagram. Red and blue squares indicate confident (Mascot score > 23 & probability of localization > 70%) and potential (Mascot score 14–22 or probability of localization 50–70%) <i>O</i>-GlcNAcylation sites, respectively. Squares surrounded in black indicate previously identified sites [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136636#pone.0136636.ref009" target="_blank">9</a>]. The HCF-1 Region II CEE amino acid sequence spanning a peptide sequence used in subsequent analyses (underlined: 901–933K) is shown below the diagram.(B) Analysis of a representative Region II CEE peptide (901–933K sequence shown in A) by LC-MS/MS for proportions of different <i>O</i>-GlcNAcylated forms. The proportions of 901–933K peptides containing 0, 1, 2 or 3 attached <i>O</i>-GlcNAc moieties are given for each sample in percent. HCF-1rep1 constructs were synthesized in HEK 293 cells and peptides were derived from constructs containing wild-type (WT) or mutated (E10A, E10D, E10Q, E10S, T17–22A) HCF-1_PRO repeats, or containing a deletion of the HCF-1_PRO-repeat sequence (∆PRO). The results with WT precursor, E10A, and E10S were confirmed in a second independent experiment.(C) HCF-1rep1 <i>O</i>-GlcNAcylation is not fundamental for HCF-1_PRO-repeat cleavage. <i>In vitro</i> cleavage activities of wild-type OGT (WT) and an <i>O</i>-GlcNAcylation compromised OGT mutant (D554H_H558D) on selected HCF-1rep1 substrates. Cleavage and <i>O</i>-GlcNAcylation activities of constructs containing the full-length HCF-1rep1 sequence (FL), or the Region II CEE (+II) or Region III (+III) sequences were analyzed by immunoblot using the indicated antibodies. We note that the lack of the OGT D554H_H558D <i>O</i>-GlcNAcylation activity results in differential mobility of the HCF-1rep1 cleavage products during electrophoresis. Prominent (●) and faint (⭕) cleavage products are indicated.</p

The Region II CEE represents an OGT-binding sequence.

<p>(A) Region II enhances OGT–HCF-1rep1 binding. Full-length (FL) and deletion HCF-1rep1 constructs were tested for OGT binding in the presence of UDP-GlcNAc using an <i>in vitro</i> OGT-directed pull-down assay. Detection of OGT and HCF-1rep1 was performed, using the indicated antibodies. Shown are 100% of OGT pull-down (panels a and b) and 11% of the input (panels c and d). *, IgG heavy chain. (B) HCF-1_PRO-repeat-independent OGT–Region II binding. (Left) Schematics of the HCF-1 constructs used in this experiment. (Right) HCF-1rep1 containing Region II and an OGT-binding defective HCF-1_PRO repeat (+II_T17–22A), or GST-fusion constructs containing Region II (wild-type or scrambled) alone or Region III alone (II_alone, II_scramb_alone, III_alone) were tested for binding with wild-type (WT) (left panel) or 5N-5A mutant (right panel) OGT. HCF-1 binding was detected as in (A). In (A) and (B), weak (⭕) and effective (●) OGT binding is indicated.</p

HCF-1_PRO-repeat cleavage enhancement by a sequence nearby the HCF-1_PRO repeat 1.

<p>(A) HCF-1_PRO-repeat cleavage is context dependent <i>in vivo</i>. (Top) Schematic of the HCF-1rep1 precursor subdivided into Region I (25 residues, blue), Region II (58 residues, pink), and Region III (60 residues, gray). (Bottom) HEK 293 cells were transfected with expression vectors encoding HCF-1rep1 FL or deletion constructs, either containing or lacking Regions I, II or III. Proteins were immunoprecipitated by an N-terminal HA-tag and assayed for cleavage by visualization by α-HA-tag immunoblot. *, C-terminal precursor truncations. (B) Region II enhances HCF-1_PRO-repeat cleavage <i>in vitro</i>. Cleavage efficiency during an <i>in vitro</i> cleavage assay time course of selected HCF-1rep1 constructs. HCF-1rep1 constructs were incubated with OGT for 0 to 8 h and precursor and resulting N-terminal cleavage products were analyzed for cleavage by α-GST-immunoblot. Uncleaved and cleaved products were quantified and cleavage efficiencies determined as cleaved products over total. Shown are the means and standard deviations of three independent experiments. (C) Region II cleavage-enhancement activity is sequence specific. <i>In vitro</i> cleavage assay of HCF-1rep1 FL and Region II constructs containing a scrambled Region II sequence (+II_scrambled) or an inactive HCF-1_PRO repeat (+II_T17–22A). Resulting precursor and N-terminal cleavage products were analyzed for cleavage with the indicated antibodies. (D) Region II activates the inactive POUrep2 construct for cleavage. (Left) Schematic of the GST-fusion construct POUrep2 containing HCF-1_PRO repeat 2 (rep2), embedded in between the POU-specific (POU_S) and POU-homeo domains (POU_H) of Oct-1. Region II or Region III were inserted N-terminal of rep2, respectively. (Right) <i>In vivo</i> cleavage activities in HEK 293 cells, transiently transfected with transfection medium (mock) or POUrep2 encoding plasmids. Precursors and cleaved fragments were purified via immunoprecipitation of an N-terminal HA-tag and cleavage assayed using the indicated antibody. In (A), (C) and (D), prominent (●) and faint (⭕) cleavage products are indicated.</p

Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT&RUN

Genome-wide localization of chromatin and transcription regulators can be detected by a variety of techniques. Here, we describe a novel method 'greenCUT&RUN' for genome-wide profiling of transcription regulators, which has a very high sensitivity, resolution, accuracy and reproducibility, whilst assuring specificity. Our strategy begins with tagging of the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. By using a GFP-nanobody the greenCUT&RUN approach eliminates antibody dependency and variability. Robust genomic profiles were obtained with greenCUT&RUN, which are accurate and unbiased towards open chromatin. By integrating greenCUT&RUN with nanobody-based affinity purification mass spectrometry, 'piggy-back' DNA binding events can be identified on a genomic scale. The unique design of greenCUT&RUN grants target protein flexibility and yields high resolution footprints. In addition, greenCUT&RUN allows rapid profiling of mutants of chromatin and transcription proteins. In conclusion, greenCUT&RUN is a widely applicable and versatile genome-mapping technique

Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT&RUN

Genome-wide localization of chromatin and transcription regulators can be detected by a variety of techniques. Here, we describe a novel method 'greenCUT&RUN' for genome-wide profiling of transcription regulators, which has a very high sensitivity, resolution, accuracy and reproducibility, whilst assuring specificity. Our strategy begins with tagging of the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. By using a GFP-nanobody the greenCUT&RUN approach eliminates antibody dependency and variability. Robust genomic profiles were obtained with greenCUT&RUN, which are accurate and unbiased towards open chromatin. By integrating greenCUT&RUN with nanobody-based affinity purification mass spectrometry, 'piggy-back' DNA binding events can be identified on a genomic scale. The unique design of greenCUT&RUN grants target protein flexibility and yields high resolution footprints. In addition, greenCUT&RUN allows rapid profiling of mutants of chromatin and transcription proteins. In conclusion, greenCUT&RUN is a widely applicable and versatile genome-mapping technique

The Palgrave Handbook of South–South Migration and Inequality

The Palgrave Handbook of South–South Migration and Inequalit

Introduction STATE OF THE CLIMATE IN 2022

Abstract —J. BLUNDEN, T. BOYER, AND E. BARTOW-GILLIES Earth’s global climate system is vast, complex, and intricately interrelated. Many areas are influenced by global-scale phenomena, including the “triple dip” La Niña conditions that prevailed in the eastern Pacific Ocean nearly continuously from mid-2020 through all of 2022; by regional phenomena such as the positive winter and summer North Atlantic Oscillation that impacted weather in parts the Northern Hemisphere and the negative Indian Ocean dipole that impacted weather in parts of the Southern Hemisphere; and by more localized systems such as high-pressure heat domes that caused extreme heat in different areas of the world. Underlying all these natural short-term variabilities are long-term climate trends due to continuous increases since the beginning of the Industrial Revolution in the atmospheric concentrations of Earth’s major greenhouse gases. In 2022, the annual global average carbon dioxide concentration in the atmosphere rose to 417.1±0.1 ppm, which is 50% greater than the pre-industrial level. Global mean tropospheric methane abundance was 165% higher than its pre-industrial level, and nitrous oxide was 24% higher. All three gases set new record-high atmospheric concentration levels in 2022. Sea-surface temperature patterns in the tropical Pacific characteristic of La Niña and attendant atmospheric patterns tend to mitigate atmospheric heat gain at the global scale, but the annual global surface temperature across land and oceans was still among the six highest in records dating as far back as the mid-1800s. It was the warmest La Niña year on record. Many areas observed record or near-record heat. Europe as a whole observed its second-warmest year on record, with sixteen individual countries observing record warmth at the national scale. Records were shattered across the continent during the summer months as heatwaves plagued the region. On 18 July, 104 stations in France broke their all-time records. One day later, England recorded a temperature of 40°C for the first time ever. China experienced its second-warmest year and warmest summer on record. In the Southern Hemisphere, the average temperature across New Zealand reached a record high for the second year in a row. While Australia’s annual temperature was slightly below the 1991–2020 average, Onslow Airport in Western Australia reached 50.7°C on 13 January, equaling Australia's highest temperature on record. While fewer in number and locations than record-high temperatures, record cold was also observed during the year. Southern Africa had its coldest August on record, with minimum temperatures as much as 5°C below normal over Angola, western Zambia, and northern Namibia. Cold outbreaks in the first half of December led to many record-low daily minimum temperature records in eastern Australia. The effects of rising temperatures and extreme heat were apparent across the Northern Hemisphere, where snow-cover extent by June 2022 was the third smallest in the 56-year record, and the seasonal duration of lake ice cover was the fourth shortest since 1980. More frequent and intense heatwaves contributed to the second-greatest average mass balance loss for Alpine glaciers around the world since the start of the record in 1970. Glaciers in the Swiss Alps lost a record 6% of their volume. In South America, the combination of drought and heat left many central Andean glaciers snow free by mid-summer in early 2022; glacial ice has a much lower albedo than snow, leading to accelerated heating of the glacier. Across the global cryosphere, permafrost temperatures continued to reach record highs at many high-latitude and mountain locations. In the high northern latitudes, the annual surface-air temperature across the Arctic was the fifth highest in the 123-year record. The seasonal Arctic minimum sea-ice extent, typically reached in September, was the 11th-smallest in the 43-year record; however, the amount of multiyear ice—ice that survives at least one summer melt season—remaining in the Arctic continued to decline. Since 2012, the Arctic has been nearly devoid of ice more than four years old. In Antarctica, an unusually large amount of snow and ice fell over the continent in 2022 due to several landfalling atmospheric rivers, which contributed to the highest annual surface mass balance, 15% to 16% above the 1991–2020 normal, since the start of two reanalyses records dating to 1980. It was the second-warmest year on record for all five of the long-term staffed weather stations on the Antarctic Peninsula. In East Antarctica, a heatwave event led to a new all-time record-high temperature of −9.4°C—44°C above the March average—on 18 March at Dome C. This was followed by the collapse of the critically unstable Conger Ice Shelf. More than 100 daily low sea-ice extent and sea-ice area records were set in 2022, including two new all-time annual record lows in net sea-ice extent and area in February. Across the world’s oceans, global mean sea level was record high for the 11th consecutive year, reaching 101.2 mm above the 1993 average when satellite altimetry measurements began, an increase of 3.3±0.7 over 2021. Globally-averaged ocean heat content was also record high in 2022, while the global sea-surface temperature was the sixth highest on record, equal with 2018. Approximately 58% of the ocean surface experienced at least one marine heatwave in 2022. In the Bay of Plenty, New Zealand’s longest continuous marine heatwave was recorded. A total of 85 named tropical storms were observed during the Northern and Southern Hemisphere storm seasons, close to the 1991–2020 average of 87. There were three Category 5 tropical cyclones across the globe—two in the western North Pacific and one in the North Atlantic. This was the fewest Category 5 storms globally since 2017. Globally, the accumulated cyclone energy was the lowest since reliable records began in 1981. Regardless, some storms caused massive damage. In the North Atlantic, Hurricane Fiona became the most intense and most destructive tropical or post-tropical cyclone in Atlantic Canada’s history, while major Hurricane Ian killed more than 100 people and became the third costliest disaster in the United States, causing damage estimated at $113 billion U.S. dollars. In the South Indian Ocean, Tropical Cyclone Batsirai dropped 2044 mm of rain at Commerson Crater in Réunion. The storm also impacted Madagascar, where 121 fatalities were reported. As is typical, some areas around the world were notably dry in 2022 and some were notably wet. In August, record high areas of land across the globe (6.2%) were experiencing extreme drought. Overall, 29% of land experienced moderate or worse categories of drought during the year. The largest drought footprint in the contiguous United States since 2012 (63%) was observed in late October. The record-breaking megadrought of central Chile continued in its 13th consecutive year, and 80-year record-low river levels in northern Argentina and Paraguay disrupted fluvial transport. In China, the Yangtze River reached record-low values. Much of equatorial eastern Africa had five consecutive below-normal rainy seasons by the end of 2022, with some areas receiving record-low precipitation totals for the year. This ongoing 2.5-year drought is the most extensive and persistent drought event in decades, and led to crop failure, millions of livestock deaths, water scarcity, and inflated prices for staple food items. In South Asia, Pakistan received around three times its normal volume of monsoon precipitation in August, with some regions receiving up to eight times their expected monthly totals. Resulting floods affected over 30 million people, caused over 1700 fatalities, led to major crop and property losses, and was recorded as one of the world’s costliest natural disasters of all time. Near Rio de Janeiro, Brazil, Petrópolis received 530 mm in 24 hours on 15 February, about 2.5 times the monthly February average, leading to the worst disaster in the city since 1931 with over 230 fatalities. On 14–15 January, the Hunga Tonga-Hunga Ha'apai submarine volcano in the South Pacific erupted multiple times. The injection of water into the atmosphere was unprecedented in both magnitude—far exceeding any previous values in the 17-year satellite record—and altitude as it penetrated into the mesosphere. The amount of water injected into the stratosphere is estimated to be 146±5 Terragrams, or ∼10% of the total amount in the stratosphere. It may take several years for the water plume to dissipate, and it is currently unknown whether this eruption will have any long-term climate effect.</jats:p