Analysis of a degron-containing reporter protein GFP-CL1 reveals a role for SUMO1 in cytosolic protein quality control.

Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential for maintaining proteostasis and normal cellular functions. Defects in PQC can result in disease, including cancer, cardiovascular disease, and neurodegeneration. The small ubiquitin-related modifiers (SUMOs) were previously implicated in the degradation of nuclear misfolded proteins, but their functions in cytoplasmic PQC are unclear. Here, in a systematic screen of SUMO protein mutations in the budding yeast Saccharomyces cerevisiae, we identified a mutant allele (Smt3-K38A/K40A) that sensitizes cells to proteotoxic stress induced by amino acid analogs. Smt3-K38A/K40A mutant strains also exhibited a defect in the turnover of a soluble PQC model substrate containing the CL1 degron (NES-GFP-Ura3-CL1) localized in the cytoplasm, but not the nucleus. Using human U2OS SUMO1- and SUMO2-KO cell lines, we observed a similar SUMO-dependent pathway for degradation of the mammalian degron-containing PQC reporter protein, GFP-CL1, also only in the cytoplasm but not the nucleus. Moreover, we found that turnover of GFP-CL1 in the cytoplasm was uniquely dependent on SUMO1 but not the SUMO2 paralogue. Additionally, we showed that turnover of GFP-CL1 in the cytoplasm is dependent on the AAA-ATPase, Cdc48/p97. Cellular fractionation studies and analysis of a SUMO1-GFP-CL1 fusion protein revealed that SUMO1 promotes cytoplasmic misfolded protein degradation by maintaining substrate solubility. Collectively, our findings reveal a conserved and previously unrecognized role for SUMO1 in regulating cytoplasmic PQC and provide valuable insights into the roles of sumoylation in PQC-associated diseases.

SUMO paralogue-specific functions revealed through systematic analysis of human knockout cell lines and gene expression data.

The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogues with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogues and also the best studied. To what extent SUMO1, SUMO2, and the other paralogues impart unique and nonredundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogues during development and studies in cell culture have relied largely on transient paralogue overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralogue gene expression levels in normal human tissues and found unique patterns of SUMO1-3 expression across 30 tissue types, suggesting paralogue-specific functions in adult human tissues. To systematically identify and characterize unique and nonredundant functions of the SUMO paralogues in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, promyelocytic leukemia (PML) nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal nonredundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.

Selection of a Clinical Lead TCR Targeting Alpha-Fetoprotein-Positive Liver Cancer Based on a Balance of Risk and Benefit.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a poor prognosis and limited therapeutic options. Alpha-fetoprotein (AFP), an established clinical biomarker of HCC, has been employed as an attractive target for T cell-based immunotherapy against this disease given its high expression in the tumor and restricted expression in normal tissues. We have identified a number of T cell receptors (TCRs) recognizing the HLA-A*02:01 restricted AFP158-166 peptide FMNKFIYEI, providing a TCR candidate pool for identifying TCRs with optimal clinical benefit. To select the ideal AFP TCR for clinical use, we evaluated the efficacy and safety profile of 7 TCRs by testing their potency toward AFP-expressing HCC cells and their specificity based upon reactivity to normal and transformed cells covering a wide variety of primary cell types and HLA serotypes. Furthermore, we assessed their cross-reactivity to potential protein candidates in the human genome by an extensive alanine scan (X-scan). We first selected three TCR candidates based on the in vitro anti-tumor activity. Next we eliminated two potential cross-reactive TCRs based on their reactivity against normal and transformed cells covering a variety of primary cell types and HLA serotypes, respectively. We then excluded the potential cross-reactivity of the selected TCR with a protein candidate identified by X-scan. At present we have selected an AFP TCR with the optimal affinity, function, and safety profile, bearing properties that are expected to allow AFP TCR redirected T cells to specifically differentiate between AFP levels on tumor and normal tissues. An early phase clinical trial using T cells transduced with this TCR to treat HCC patients (NCT03971747) has been initiated.

Short- and long-term use of medication for psychological distress after the diagnosis of cancer.

PURPOSE: This study investigated the short- and long-term use of medication for psychological distress after the diagnosis of cancer. METHODS: Longitudinal data from the Taiwan National Health Insurance database were used to follow 35,137 cancer patients for 2.5 years after being diagnosed in 2006 and 2007. RESULTS: Among those patients who survived for at least 180 days, 20.9 % had used psychotropic medications; sedatives were the most frequently prescribed (14.3 %), followed by antidepressants (5.5 %), anxiolytics (3.6 %), and antipsychotics (2.7 %). Lung cancer, prostate cancer, and oral cancer showed a significant association with the regular use of medication in the first 180 days. Among patients who survived for at least 2.5 years, 4.8 % still used psychotropic medication on a regular basis. Lung cancer and prostate cancer were associated with such prolonged use. CONCLUSIONS: This longitudinal study found that the type of cancer was significantly associated with the use of psychotropic drugs after the diagnosis was made. It provided information about the trajectory of that use and found that a small number of patients were still using those medications after 2.5 years.

Genetic variants of IL-6 and its receptor are not associated with schizophrenia in Taiwan.

The pathophysiological process of schizophrenia is still unclear. The levels of interleukine-6 (IL-6) and its receptor, soluble IL-6R, have been reported to be elevated in the plasma and cerebrospinal fluid of schizophrenic patients. In this study, we tested the association of genetic variants of IL-6 and IL-6R with schizophrenia. Genotyping of three single nucleotide polymorphisms (SNP) for each IL-6 (IL-6-1, IL-6-2, and IL-6-3) and IL-6R (rs4845617=IL-6R1, rs4553185=IL-6R2, and rs4379670=IL-6R3) gene was performed in 100 patients with schizophrenia and 113 normal controls. The polymorphisms of IL-6R2 were genotyped using Tetra-primer ARMS PCR. IL-6R3 polymorphisms were genotyped using restriction fragment length polymorphism (RFLP) with Apo I enzyme as the restriction enzyme. All other polymorphisms were genotyped using the direct sequencing method. We found a di-nucleotide haplotype block and a tri-nucleotide haplotype block in the genes of IL-6 and IL-6R, respectively. All six SNPs and their haplotypes failed to show a significant association with schizophrenia. The IL-6-2 SNP showed a nominally significant association with the positive symptoms of schizophrenia (p=0.0472). We conclude that the genetic variants of IL-6 and IL-6R are not associated with schizophrenia. In order to verify this result, further study using a larger sample size and exploring the association between the genotype of IL-6-2 and plasma level of IL-6 is recommended.

The influences of Taiwan's National Health Insurance on women's choice of prenatal care facility: Investigation of differences between rural and non-rural areas.

BACKGROUND: Taiwan's National Health Insurance (NHI), implemented in 1995, substantially increased the number of health care facilities that can deliver free prenatal care. Because of the increase in such facilities, it is usually assumed that women would have more choices regarding prenatal care facilities and thus experience reduction in travel cost. Nevertheless, there has been no research exploring these issues in the literature. This study compares how Taiwan's NHI program may have influenced choice of prenatal care facility and perception regarding convenience in transportation for obtaining such care for women in rural and non-rural areas in Taiwan. METHODS: Based on data collected by a national survey conducted by Taiwan's National Health Research Institutes (NHRI) in 2000, we tried to compare how women chose prenatal care facility before and after Taiwan's National Health Insurance program was implemented. Basing our analysis on how women answered questionnaire items regarding "the type of major health care facility used and convenience of transportation to and from prenatal care facility," we investigated whether there were disparities in how women in rural and non-rural areas chose prenatal care facilities and felt about the transportation, and whether the NHI had different influences for the two groups of women. RESULTS: After NHI, women in rural areas were more likely than before to choose large hospitals for prenatal care services. For women in rural areas, the relative probability of choosing large hospitals to choosing non-hospital settings in 1998-1999 was about 6.54 times of that in 1990-1992. In contrast, no such change was found in women in non-rural areas. For a woman in a non-rural area, she was significantly more likely to perceive the transportation to and from prenatal care facilities to be very convenient between 1998 and 1999 than in the period between 1990 and 1992. No such improvement was found for women in rural areas. CONCLUSION: We concluded that women in rural areas were more likely to seek prenatal care in large hospitals, but were not more likely to perceive very convenient transportation to and from prenatal care facilities in the late 1990s than in the early 1990s. In contrast, women in non-rural areas did not have a stronger tendency to seek prenatal care in large hospitals in the late 1990s than in earlier periods. In addition, they did perceive an improvement in transportation for acquiring prenatal care in the late 1990s. More efforts should be made to reduce these disparities.

An investigation of the smoking behaviours of parents before, during and after the birth of their children in Taiwan.

BACKGROUND: Although many studies have investigated the negative effects of parental smoking on children and Taiwan has started campaigns to promote smoke-free homes, little is known about the smoking behaviours of Taiwanese parents during the childbearing period. To help fill the gap, this study investigated Taiwanese parents' smoking behaviours before, during and after the birth of their children, particularly focusing on smoking cessation during pregnancy and relapse after childbirth. METHODS: We used data from the Survey of Health Status of Women and Children, conducted by Taiwan's National Health Research Institutes in 2000. After excluding survey respondents with missing information about their smoking behaviours, our sample consisted of 3,109 women who were married at the time of interview and had at least one childbearing experience between March 1, 1995 and February 28, 1999. Data on parental smoking behaviour in the six months before pregnancy, during pregnancy, and in the first year after childbirth were extracted from the survey and analysed by descriptive statistics as well as logistic regression. RESULTS: Four percent of the mothers and sixty percent of the fathers smoked before the conception of their first child. The educational attainment and occupation of the parents were associated with their smoking status before the first pregnancy in the family. Over 80% of smoking mothers did not quit during pregnancy, and almost all of the smoking fathers continued tobacco use while their partners were pregnant. Over two thirds of the women who stopped smoking during their pregnancies relapsed soon after childbirth. Very few smoking men stopped tobacco use while their partners were pregnant, and over a half of those who quit started to smoke again soon after their children were born. CONCLUSION: Among Taiwanese women who had childbearing experiences in the late 1990s, few smoked. Of those who smoked, few quit during pregnancy. Most of those who quit relapsed in the first year after childbirth. The smoking prevalence was high among the husbands of these Taiwanese women, and almost all of these smoking fathers continued tobacco use while their partners were pregnant. It is important to advocate the benefits of a smoke-free home to Taiwanese parents-to-be and parents with young children, especially the fathers. The government should take advantage of its free prenatal care and well-child care services to do this. In addition to educational campaigns through the media, the government can request physicians to promote smoke-free homes when they deliver prenatal care and well-child care. This could help reduce young children's health risks from their mothers' smoking during pregnancy and second-hand smoke at home.