Michal Bassani-Sternberg
Mar 1, 2015; 14:658-673
Research

Hasmik Keshishian
Dec 1, 2007; 6:2212-2229
Research

Sean R. Collins
Mar 1, 2007; 6:439-450
Research

Mathias Uhlén
Dec 1, 2005; 4:1920-1932
Research

Leigh Anderson
Apr 1, 2006; 5:573-588
Research

N. Leigh Anderson
Nov 1, 2002; 1:845-867
Reviews/Perspectives

Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2–depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3β (GSK3β) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine–ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure–function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase–ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.

In Staphylococcus aureus–caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1–325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1–246) as a probe for competitively defining the affinities of N-terminal SC(1–246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1–246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1–246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1–246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1–246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.

The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.

Members Event Webinar

Event participants

Pepijn Bergsen, Research Fellow, Europe Programme, Chatham House

Dr Sheri Berman, Professor of Political Science, Barnard College

Chair: Hans Kundnani, Senior Research Fellow, Europe Programme, Chatham House

Nica M. Borradaile
Dec 1, 2006; 47:2726-2737
Research Articles

Margrit Schwarz
Sep 1, 1998; 39:1833-1843
Articles

Oswald Quehenberger
Nov 1, 2010; 51:3299-3305
Research Articles

WS Harris
Jun 1, 1989; 30:785-807
Reviews

Ronald M. Krauss
Jan 1, 1982; 23:97-104
Articles

AR Tall
Aug 1, 1993; 34:1255-1274
Reviews

G Lepage
Jan 1, 1986; 27:114-120
Articles

John A. Glomset
Mar 1, 1968; 9:155-167
Reviews

Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behcet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position ). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to ~40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.

Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (~3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.

A. A. Vladimirov
Trans. Moscow Math. Soc. 80 (2020), 211-219.
Abstract, references and article information

A. G. Sergeev and Kh. A. Khachatryan
Trans. Moscow Math. Soc. 80 (2020), 95-111.
Abstract, references and article information

S. Yu. Orevkov
Trans. Moscow Math. Soc. 80 (2020), 73-81.
Abstract, references and article information

THERE'S nothing like the sweet taste of victory, especially when it's long in coming.

Shukai Du and Francisco-Javier Sayas
Math. Comp. 89 (2019), 1745-1782.
Abstract, references and article information

Cătălin I. Cârstea, Gen Nakamura and Lauri Oksanen
Trans. Amer. Math. Soc. 373 (2020), 3423-3443.
Abstract, references and article information

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Airfix 1:72 RNLI Severn Class - Plymouth Lifeboat

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Carla Farsi, Laura Scull and Jordan Watts
Proc. Amer. Math. Soc. 148 (2020), 2717-2737.
Abstract, references and article information

Bin Li and Jieqiong Shen
Proc. Amer. Math. Soc. 148 (2020), 2565-2578.
Abstract, references and article information

Naoya Hiramatsu and Ryo Takahashi
Proc. Amer. Math. Soc. 148 (2020), 2359-2369.
Abstract, references and article information

Chuanan Wei
Proc. Amer. Math. Soc. 148 (2020), 2287-2296.
Abstract, references and article information

The Security Bureau today said that the last Hong Kong resident who had contracted COVID-19 while aboard the Diamond Princess cruise ship and was hospitalised in Japan has returned to Hong Kong.

Immigration Department staff that assisted Hong Kong residents in Japan have completed their mission and returned to Hong Kong as well.

In early February, a cluster of COVID-19 infection cases occurred on the Diamond Princess cruise docked in Yokohama.

Of some 3,700 passengers and crew, about 370 were from Hong Kong. The 712 confirmed COVID-19 cases associated with the cruise included 76 Hong Kong residents who were hospitalised in Japan for isolation and treatment.

While three Hong Kong residents passed away, the remaining 73 patients returned to Hong Kong or their places of residence after being discharged from the hospital.

As for other Hong Kong residents on the cruise, the bureau noted that the Hong Kong Special Administrative Region Government arranged three flights, between February 19 and 23, to escort 193 of them back to the city.

Upon arrival, they were transferred to the quarantine centre at Chun Yeung Estate to undergo 14 days of quarantine.

Another 144 Hong Kong residents returned on their own via other flights, including 25 close contacts of the patients who had completed quarantine in Japan.

For those who returned to Hong Kong on their own and did not complete 14 days of quarantine in Japan, they were required to complete the remaining quarantine period at a quarantine centre.

Of the 231 cruise passengers admitted to the quarantine centre at Chun Yeung Estate, nine tested positive for COVID-19 and were sent to hospitals for isolation and treatment.

The Hong Kong SAR Government expressed profound condolences on the passing of the Hong Kong patients and its deepest sympathies to their families.

The SAR Government emphasised that the incident could not have been resolved smoothly without the staunch support of the Office of the Commissioner of the Ministry of Foreign Affairs of the People's Republic of China in the Hong Kong Special Administrative Region, the Embassy of the People’s Republic of China in Japan and Japanese authorities.

The SAR Government also thanked Cathay Pacific Airways and the Airport Authority for their assistance, the bureau added.

The partnership will join hands with four UK startups as it hopes to reduce plastic waste as part of its retail tech initiative, JLAB