Centromere/kinetochore is assembled through CENP-C oligomerization.

Kinetochore is an essential protein complex required for accurate chromosome segregation. The constitutive centromere-associated network (CCAN), a subcomplex of the kinetochore, associates with centromeric chromatin and provides a platform for the kinetochore assembly. The CCAN protein CENP-C is thought to be a central hub for the centromere/kinetochore organization. However, the role of CENP-C in CCAN assembly needs to be elucidated. Here, we demonstrate that both the CCAN-binding domain and the C-terminal region that includes the Cupin domain of CENP-C are necessary and sufficient for chicken CENP-C function. Structural and biochemical analyses reveal self-oligomerization of the Cupin domains of chicken and human CENP-C. We find that the CENP-C Cupin domain oligomerization is vital for CENP-C function, centromeric localization of CCAN, and centromeric chromatin organization. These results suggest that CENP-C facilitates the centromere/kinetochore assembly through its oligomerization.

Investigation of the incidence of immunisation stress-related response following COVID-19 vaccination in healthcare workers.

INTRODUCTION: Safe vaccination worldwide is critical to end the coronavirus disease 2019 (COVID-19) pandemic. We aimed to evaluate adverse reactions to vaccination using a web-based questionnaire and examine the risk factors for the occurrence of immunisation stress-related response (ISRR). METHODS: We conducted a questionnaire survey using Google Form® among the employees of St. Marianna University Hospital who had received the COVID-19 vaccine between April 2021 and May 2021, 1 week after the first and second vaccinations. We developed and used a questionnaire to identify individuals with ISRR according to the World Health Organization diagnostic criteria. A generalised linear mixed model was constructed with ISRR onset as the dependent variable, subjects as the random factor, and each parameter as a fixed factor. A multivariate model was constructed using the forced imputation method with factors that were significant in the univariate analysis. RESULTS: We enrolled 2,073 and 1,856 respondents in the first and second questionnaire surveys, respectively. Fifty-five and 33 ISRR cases were identified in the first and second vaccinations, respectively. In the univariate analysis, strong pre-vaccination anxiety (odds ratio [OR], 2.3; 95% confidence interval [CI], 1.30-4.12, p = 0·004) and history of allergy (OR, 1.6; 95% CI, 1.14-2.24, p = 0·007) were significant risk factors. Multivariate analysis also showed that strong pre-vaccination anxiety (OR, 2.1; 95% CI, 1.15-3.80, p = 0.016) and history of allergy (OR, 1.5; 95% CI, 1.09-2.15, p = 0.014) were significant risk factors. CONCLUSIONS: Confirmation of allergy prior to vaccination and subsequent action are essential for addressing ISRR.

Recruitment of two Ndc80 complexes via the CENP-T pathway is sufficient for kinetochore functions.

To form functional kinetochores, CENP-C and CENP-T independently recruit the KMN (Knl1C, Mis12C, and Ndc80C) network onto the kinetochores. To clarify the functions of the KMN network on CENP-T, we evaluated its roles in chicken DT40 cell lines lacking the CENP-C-KMN network interaction. By analyzing mutants lacking both CENP-T-Mis12C and CENP-C-Mis12C interactions, we demonstrated that Knl1C and Mis12C (KM) play critical roles in the cohesion of sister chromatids or the recruitment of spindle checkpoint proteins onto kinetochores. Two copies of Ndc80C (N-N) exist on CENP-T via Mis12C or direct binding. Analyses of cells specifically lacking the Mis12C-Ndc80C interaction revealed that N-N is needed for proper kinetochore-microtubule interactions. However, using artificial engineering to directly bind the two copies of Ndc80C to CENP-T, we demonstrated that N-N functions without direct Mis12C binding to Ndc80C in native kinetochores. This study demonstrated the mechanisms by which complicated networks play roles in native kinetochores.

Mobility of kinetochore proteins measured by FRAP analysis in living cells.

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

The GNU subunit of PNG kinase, the developmental regulator of mRNA translation, binds BIC-C to localize to RNP granules.

Control of mRNA translation is a key mechanism by which the differentiated oocyte transitions to a totipotent embryo. In Drosophila, the PNG kinase complex regulates maternal mRNA translation at the oocyte-to-embryo transition. We previously showed that the GNU activating subunit is crucial in regulating PNG and timing its activity to the window between egg activation and early embryogenesis (Hara et al., 2017). In this study, we find associations between GNU and proteins of RNP granules and demonstrate that GNU localizes to cytoplasmic RNP granules in the mature oocyte, identifying GNU as a new component of a subset of RNP granules. Furthermore, we define roles for the domains of GNU. Interactions between GNU and the granule component BIC-C reveal potential conserved functions for translational regulation in metazoan development. We propose that by binding to BIC-C, upon egg activation GNU brings PNG to its initial targets, translational repressors in RNP granules.

Non-invasive fibrosis assessments of non-alcoholic fatty liver disease associated with low estimated glomerular filtration rate among CKD patients: the Fukuoka Kidney disease Registry Study.

BACKGROUND: A growing body of evidence has shown that non-alcoholic fatty liver disease (NAFLD) is associated with chronic kidney disease (CKD). Non-invasive fibrosis assessments of NAFLD such as Fibrosis-4 (FIB-4) index and NAFLD fibrosis score (NFS) have been developed to substitute liver biopsy. Little is known about the association between FIB-4 index or NFS and the components of CKD. METHODS: In the present cross-sectional study, we assessed of 3640 Japanese CKD patients. We examined the association between FIB-4index or NFS and the odds of having low estimated glomerular filtration rate (eGFR) defined as eGFR < 60 mL/min/1.73 m2 or albuminuria defined as urinary albumin-to-creatinine ratio (UACR) ≥ 30 mg/g. Patients were divided into quartiles according to their baseline FIB-4 index and NFS levels. Linear and logistic regression analysis were conducted, with adjustment for potential confounding factors. RESULTS: FIB-4 index and NFS were negatively associated with eGFR, but not UACR, after adjustment for potential confounding factors. Both FIB-4 index and NFS were significantly associated with low eGFR after adjustment for potential confounding factors. Meanwhile, in the multivariable-adjusted model, no associations were found between FIB-4 index or NFS and albuminuria. The addition of FIB-4 index or NFS to the established clinical CKD risk factors improved diagnostic accuracy of prevalence of low eGFR. We also found that there was a significant trend of higher FIB-4 index and NFS with more advanced renal fibrosis using the kidney biopsy data. CONCLUSIONS: Higher non-invasive fibrosis assessments of NAFLD were associated with higher odds of decreased eGFR.

CENP-C Phosphorylation by CDK1 in vitro.

Accurate chromosome segregation during mitosis requires the kinetochore, a large protein complex, which makes a linkage between chromosomes and spindle microtubes. An essential kinetochore component, CENP-C, is phosphorylated by Cyclin-B-Cyclin dependent kinase 1 (CDK1) that is a master kinase for mitotic progression, promoting proper kinetochore assembly during mitosis. Here, we describe an in vitro CDK1 kinase assay to detect CENP-C phosphorylation using Phos-tag SDS-PAGE without radiolabeled ATP. Our protocol has advantages in ease and safety over conventional phosphorylation assays using [γ-32P]-ATP, which has potential hazards despite their better sensitivity. The protocol described here can be applicable to other kinases and be also useful for analysis of phospho-sites in substrates in vitro.

Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C.

The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.

Arginase 2 is a mediator of ischemia-reperfusion injury in the kidney through regulation of nitrosative stress.

Kidney ischemia-reperfusion injury is a major cause of acute kidney injury (AKI). Following reduced kidney perfusion, the pathological overproduction of reactive oxygen and reactive nitrogen species play a substantial role in the development of kidney ischemia-reperfusion injury. Arginase 2 (ARG2) competes with nitric oxide synthase for the same substrate, L-arginine, and is implicated in the regulation of reactive nitrogen species. Therefore, we investigated the role of ARG2 in kidney ischemia-reperfusion injury using human proximal tubule cells (HK-2) and a mouse model of kidney ischemia-reperfusion injury. ARG2 was predominantly expressed in kidney tubules of the cortex, which was increased after ischemia-reperfusion injury. In HK-2 cells, ARG2 was expressed in punctate form in the cytoplasm and upregulated after hypoxia-reoxygenation. ARG2 knockdown reduced the level of reactive oxygen species and 3-nitrotyrosine after hypoxia-reoxygenation injury compared with control siRNA. Consistent with these results, in Arg2 knockout mice, abnormal kidney function and the increased acute tubular necrosis score induced by ischemia-reperfusion injury was significantly reduced without any obvious blood pressure changes. Additionally, an accumulation of 3-nitrotyrosine and apoptosis of renal tubule cells were attenuated in Arg2 knockout mice compared with wild-type mice. Inhibition of arginase by Nω-hydroxy-nor-L-arginine alleviated kidney ischemia-reperfusion injury like the results found in Arg2 knockout mice. Thus, ARG2 plays a pivotal role in ischemia-reperfusion-induced AKI by means of nitrosative stress. Hence, an ARG2-specific inhibitor may effectively treat kidney ischemia-reperfusion injury.

The Association between Dialysis Dose and Risk of Cancer Death in Patients Undergoing Hemodialysis: The Q-Cohort Study.

Objective Uremic toxins are known risk factors for cancer in patients undergoing hemodialysis (HD). Although adequate removal of uremic toxins might reduce the cancer risk by improving subclinical uremia, the relationship between the dialysis dose and risk of cancer death in patients undergoing HD remains unclear. Methods In this prospective observational study, 3,450 patients undergoing HD were followed up for 4 years. The primary outcome was cancer death. Patients were divided into quartiles according to their baseline Kt/V levels. The association between the Kt/V levels and risk of cancer death was estimated using the Kaplan-Meier method and Cox proportional-hazards model. Results A total of 111 patients (3.2%) died from cancer during the 4-year observational period. The 4-year survival rate decreased linearly with decreasing Kt/V. The multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for cancer death were 2.23 (95% CI, 1.13-4.56), 1.77 (0.88-3.63), and 1.89 (1.04-3.56) in quartile (Q) 1, Q2, and Q3, respectively, compared with patients in the highest Kt/V category (Q4) (p for trend = 0.06). Every 0.1 increase in Kt/V was associated with a reduction of 8% in cancer death (HR 0.92, 95% CI, 0.85-0.99). Conclusion A lower dialysis dose might be associated with a higher risk of cancer death in patients undergoing HD. Kt/V is a simple indicator of dialysis dose used in clinical practice and might be a useful modifiable factor for predicting the risk of cancer death. Further basic and interventional studies are needed to confirm the apparent reduction in cancer death associated with increasing the dialysis dose.

Dynamics of kinetochore structure and its regulations during mitotic progression.

Faithful chromosome segregation during mitosis in eukaryotes requires attachment of the kinetochore, a large protein complex assembled on the centromere of each chromosome, to the spindle microtubules. The kinetochore is a structural interface for the microtubule attachment and provides molecular surveillance mechanisms that monitor and ensure the precise microtubule attachment as well, including error correction and spindle assembly checkpoint. During mitotic progression, the kinetochore undergoes dynamic morphological changes that are observable through electron microscopy as well as through fluorescence microscopy. These structural changes might be associated with the kinetochore function. In this review, we summarize how the dynamics of kinetochore morphology are associated with its functions and discuss recent findings on the switching of protein interaction networks in the kinetochore during cell cycle progression.

Association of Lower Serum Bilirubin With Loss of Residual Kidney Function in Peritoneal Dialysis Patients.

Bilirubin is recognized as an endogenous antioxidant, and low serum bilirubin is reported to be associated with the progression of kidney disease. However, it is unclear whether serum bilirubin levels are associated with the loss of residual kidney function (RKF) in peritoneal dialysis (PD) patients. This study investigated the relationship between serum total bilirubin and loss of RKF. We prospectively followed 94 PD patients who started PD in our hospital between June 2006 and May 2016. Ten patients who had chronic liver disease or cirrhosis were excluded. Patients were divided into three groups based on serum total bilirubin concentration tertiles: tertile 1 (T1) < 0.3, T2 = 0.3, and T3 ≥ 0.4 mg/dL. We estimated the relationship between serum bilirubin and loss of RKF, defined as daily urine volume (<100 mL) within 3 years after starting PD, using a Cox proportional hazards model. During the 3-year observation period, 22 patients lost RKF. The incidence rate of loss of RKF increased linearly with the decrease in serum total bilirubin levels (P for trend < 0.05). After adjusting for confounding factors, low serum total bilirubin level was shown to be an independent predictor of loss of RKF (hazard ratio [HR] for every 0.1 mg/dL decrease, 1.50; 95% confidence interval [CI], 1.01-2.51; HR [95%CI] for T2 and T1 [vs. T3] 2.03 [0.65-7.88] and 3.70 [1.00-15.9]). This study suggests that low serum total bilirubin levels are associated with the loss of RKF in PD patients.

CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization.

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A-CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A-binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A-CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.

Where is the right path heading from the centromere to spindle microtubules?

The kinetochore is a large protein complex that ensures accurate chromosome segregation during mitosis by connecting the centromere and spindle microtubules. One of the kinetochore sub-complexes, the constitutive centromere-associated network (CCAN), associates with the centromere and recruits another sub-complex, the KMN (KNL1, Mis12, and Ndc80 complexes) network (KMN), which binds to spindle microtubules. The CCAN-KMN interaction is mediated by two parallel pathways (CENP-C- and CENP-T-pathways) in the kinetochore, which bridge the centromere and microtubules. Here, we discuss dynamic protein-interaction changes in the two pathways that couple the centromere with spindle microtubules during mitotic progression.

Association of B-Type Natriuretic Peptide Level With Residual Kidney Function in Incident Peritoneal Dialysis Patients.

BACKGROUND: Residual kidney function (RKF) is an important factor influencing both technique and patient survival in peritoneal dialysis (PD) patients. B-type natriuretic peptide (BNP) is considered a marker of cardio-renal syndrome. The relationship between BNP and RKF in PD patients remains unclear. METHODS: We conducted a prospective study of 89 patients who had started and continued PD for 6 months or more in Kyushu University Hospital between June 2006 and September 2015. Participants were divided into low BNP (≤ 102.1 ng/L) and high BNP (> 102.1 ng/L) groups according to median plasma BNP level at PD initiation. The primary outcome was RKF loss, defined as 24-hour urine volume less than 100 mL. We estimated the association between BNP and RKF loss using a Kaplan-Meier method and Cox proportional hazards model and compared the rate of RKF decline between the 2 groups. To evaluate the consistency of the association, we performed subgroup analysis stratified by baseline characteristics. RESULTS: During the median follow-up of 30 months, 30 patients lost RKF. Participants in the high BNP group had a 5.87-fold increased risk for RKF loss compared with the low BNP group after adjustment for clinical and cardiac parameters. A high plasma BNP level was more clearly associated with RKF loss in younger participants compared with older participants in subgroup analysis. CONCLUSIONS: B-type natriuretic peptide may be a useful risk marker for RKF loss in PD patients. The clinical importance of plasma BNP level as a marker of RKF loss might be affected by age.

Prognostic value of pre-dialysis blood pressure and risk threshold on clinical outcomes in hemodialysis patients: The Q-Cohort Study.

The influence of pre-dialysis blood pressure (BP) on the prognosis of hemodialysis (HD) patients is still inconclusive.A total of 3436 HD patients were prospectively followed up for 4 years. The patients were divided into quintiles of pre-dialysis systolic BP (SBP) and diastolic BP (DBP) levels [mm Hg]: Quintile 1 (Q1), SBP <134, DBP <66; Q2, SBP 134 to 147, DBP 66 to 72; Q3, SBP 148 to 158, DBP 73 to 79; Q4, SBP 159 to 171, DBP 80 to 85; Q5, SBP ≥172, DBP ≥86. The association between the pre-dialysis BP and outcomes were examined using a Cox proportional hazards model.During a 4-year follow-up period, 564 (16.4%) patients died of any cause and 590 (17.2%) developed cardiovascular (CV) events. The lowest level of pre-dialysis SBP group (Q1) showed a significantly increased risk of all-cause mortality (hazard ratio [HR] 1.83, 95% confidence interval [CI] 1.40-2.39) and the highest group (Q5) significantly increased risk of CV events (HR 1.31, 95% CI 1.02-1.68) compared with the reference group (Q3), respectively. The highest level of pre-dialysis DBP group was significantly associated with increased risk for both all-cause mortality and CV events. Restricted cubic spline analysis for BP and outcomes suggested the optimal pre-dialysis BP value associated with the lowest risk of outcomes was SBP 152 mm Hg for all-cause mortality, SBP 143 mm Hg for CV events, and DBP 68 mm Hg for all-cause mortality.Our results suggested that pre-dialysis BP was independently associated with all-cause mortality and CV events among Japanese HD patients.

Publisher Correction: Multiple phosphorylations control recruitment of the KMN network onto kinetochores.

In the version of this Article originally published, the 'ON' and 'OFF' labels in panel c of Fig. 6 were incorrect. For the Tet treated cells (+Tet) in both image panels, CENP-T should have been 'OFF' and CENP-T Δ90 should have been 'ON'. For the cells untreated with Tet (-Tet) in both graph panels, CENP-T Δ90 should have been 'ON'. This has now been amended.

Multiple phosphorylations control recruitment of the KMN network onto kinetochores.

To establish a functional kinetochore, the constitutive centromere-associated network (CCAN) forms a foundation on the centromere and recruits the KMN network, which directly binds to spindle microtubules. The CENP-C and CENP-T pathways in the CCAN recruit the KMN network to kinetochores, independently. The CENP-C pathway has been considered the major scaffold for the KMN network in vertebrate CCAN. However, we demonstrate that it is mainly the CENP-T pathway that recruits the KMN network onto the kinetochores and that CENP-T-KMN interactions are essential in chicken DT40 cells. By contrast, less Ndc80 binds to the CENP-C pathway in mitosis and the Mis12-CENP-C association is decreased during mitotic progression, which is consistent with the finding that the Mis12 complex-CENP-C binding is dispensable for cell viability. Furthermore, we find that multiple phosphoregulations of CENP-T and the Mis12 complex make the CENP-T pathway dominant. These results provide key insights into kinetochore dynamics during mitotic progression.

Kinetochore assembly and disassembly during mitotic entry and exit.

Faithful chromosome segregation during mitosis in eukaryotes requires a large protein complex, kinetochore, formed on the centromere of each chromosome, to attach to spindle microtubules. Among the kinetochore proteins, Constitutive Centromere-Associated Network (CCAN) and KMN-network proteins form the base of the vertebrate kinetochore architecture. The CCAN proteins constitutively localize to the centromere throughout the cell cycle, whereas KMN-network proteins are recruited to the CCAN only during mitosis. Recent studies in cellular and structural biology, as well as biochemical reconstitutions, have revealed that mitotic phosphorylation of kinetochore proteins has critical roles in kinetochore organization. Here, we discuss the molecular processes of kinetochore assembly during mitotic entry and its disassembly during mitotic exit.