The two products of the KRAS locus, K-Ras4A and K-Ras4B, are encoded by alternative fourth exons and therefore, possess distinct membrane-targeting sequences. The common activating mutations occur in exons 1 or 2 and therefore, render both splice variants oncogenic. K-Ras4A has been understudied, because it has been considered a minor splice variant. By priming off of the splice junction, we developed a quantitative RT-PCR assay for K-Ras4A and K-Ras4B message capable of measuring absolute amounts of the two transcripts. We found that K-Ras4A was widely expressed in 30 of 30 human cancer cell lines and amounts equal to K-Ras4B in 17 human colorectal tumors. Using splice variant-specific antibodies, we detected K-Ras4A protein in several tumor cell lines at a level equal to or greater than that of K-Ras4B. In addition to the CAAX motif, the C terminus of K-Ras4A contains a site of palmitoylation as well as a bipartite polybasic region. Although both were required for maximal efficiency, each of these could independently deliver K-Ras4A to the plasma membrane. Thus, among four Ras proteins, K-Ras4A is unique in possessing a dual membrane-targeting motif. We also found that, unlike K-Ras4B, K-Ras4A does not bind to the cytosolic chaperone δ-subunit of cGMP phosphodiesterase type 6 (PDE6δ). We conclude that efforts to develop anti–K-Ras drugs that interfere with membrane trafficking will have to take into account the distinct modes of targeting of the two K-Ras splice variants.

Erv1 is a flavin‐dependent sulfhydryl oxidase in the mitochondrial intermembrane space (IMS) that functions in the import of cysteine‐rich proteins. Redox titrations of recombinant Erv1 showed that it contains three distinct couples with midpoint potentials of −320, −215, and −150 mV. Like all redox‐active enzymes, Erv1 requires one or more electron acceptors. We have generated strains with erv1 conditional alleles and employed biochemical and genetic strategies to facilitate identifying redox pathways involving Erv1. Here, we report that Erv1 forms a 1:1 complex with cytochrome c and a reduced Erv1 can transfer electrons directly to the ferric form of the cytochrome. Erv1 also utilized molecular oxygen as an electron acceptor to generate hydrogen peroxide, which is subsequently reduced to water by cytochrome c peroxidase (Ccp1). Oxidized Ccp1 was in turn reduced by the Erv1‐reduced cytochrome c. By coupling these pathways, cytochrome c and Ccp1 function efficiently as Erv1‐dependent electron acceptors. Thus, we propose that Erv1 utilizes diverse pathways for electron shuttling in the IMS.

SUMMARY A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin and cycloheximide, increase steady-state palmitoylation. FK506 inhibits retrograde trafficking of H-Ras from the plasma membrane to the Golgi in a proline 179-dependent fashion, augments early GTP-loading of Ras in response to growth factors, and promotes H-Ras dependent neurite outgrowth from PC12 cells. These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines.

Background: Racial inequities for patients with heart failure (HF) have been widely documented. HF patients who receive cardiology care during a hospital admission have better outcomes. It is unknown whether there are differences in admission to a cardiology or general medicine service by race. This study examined the relationship between race and admission service, and its effect on 30-day readmission and mortality Methods: We performed a retrospective cohort study from September 2008 to November 2017 at a single large urban academic referral center of all patients self-referred to the emergency department and admitted to either the cardiology or general medicine service with a principal diagnosis of HF, who self-identified as white, black, or Latinx. We used multivariable generalized estimating equation models to assess the relationship between race and admission to the cardiology service. We used Cox regression to assess the association between race, admission service, and 30-day readmission and mortality. Results: Among 1967 unique patients (66.7% white, 23.6% black, and 9.7% Latinx), black and Latinx patients had lower rates of admission to the cardiology service than white patients (adjusted rate ratio, 0.91; 95% CI, 0.84–0.98, for black; adjusted rate ratio, 0.83; 95% CI, 0.72–0.97 for Latinx). Female sex and age >75 years were also independently associated with lower rates of admission to the cardiology service. Admission to the cardiology service was independently associated with decreased readmission within 30 days, independent of race. Conclusions: Black and Latinx patients were less likely to be admitted to cardiology for HF care. This inequity may, in part, drive racial inequities in HF outcomes.

Significance K-Ras is mutated more often than any other oncogene, making the protein and the pathways it regulates attractive targets for anticancer drug discovery. We have shown that phosphorylation of serine 181 in the membrane-targeting region of K-Ras causes the protein to translocate from plasma membrane to intracellular membranes. Translocation is associated with toxicity but the mechanism has remained undefined. Here we show that phospho–K-Ras associates with inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER) and thereby blocks one of the prosurvival activities of Bcl-xL, which is to sensitize IP3Rs and thereby allow constitutive transfer of calcium from ER to mitochondria where it is required for efficient respiration. This pathway could be exploited to limit the oncogenic activity of mutant K-Ras.

Inherited bone marrow failure syndromes (IBMFS) are characterized by ineffective hematopoiesis and increased risk of developing myeloid malignancy. The pathophysiologies of different IBMFS are variable, and can relate to defects in diverse biological processes, including DNA damage repair (Fanconi anemia), telomere maintenance (dyskeratosis congenita), and ribosome biogenesis (Diamond-Blackfan anemia, Shwachman-Diamond syndrome). Somatic mutations leading to clonal hematopoiesis have been described in IBMFS, but the distinct mechanisms by which mutations drive clonal advantage in each disease and their associations with leukemia risk are not well understood. Clinical observations and laboratory models of IBMFS suggest that the germline deficiencies establish a qualitatively impaired functional state at baseline. In this context, somatic alterations can promote clonal hematopoiesis by improving the competitive fitness of specific hematopoietic stem cell clones. Some somatic alterations relieve baseline fitness constraints by normalizing the underlying germline deficit through direct reversion or indirect compensation, while others do so by subverting senescence or tumor suppressor pathways. Clones with normalizing somatic mutations may have limited transformation potential due to retention of functionally intact fitness-sensing and tumor suppressor pathways, while those with mutations that impair cellular elimination may have increased risk of malignant transformation due to subversion of tumor suppressor pathways. Since clonal hematopoiesis is not deterministic of malignant transformation, rational surveillance strategies will thus depend on the ability to prospectively identify specific clones with increased leukemic potential. We describe a framework by which an understanding of the processes that promote clonal hematopoiesis in IBMFS may inform clinical surveillance strategies.

Germline pathogenic TERT variants are associated with short telomeres and an increased risk of developing myelodysplastic syndrome (MDS) among patients with a telomere biology disorder. We identified TERT rare variants in 41 of 1514 MDS patients (2.7%) without a clinical diagnosis of a telomere biology disorder who underwent allogeneic transplantation. Patients with a TERT rare variant had shorter telomere length (p<0.001) and younger age at MDS diagnosis (52 vs. 59 years, p=0.03) than patients without a TERT rare variant. In multivariable models, TERT rare variants were associated with inferior overall survival (p=0.034) driven by an increased incidence of non-relapse mortality (NRM) (p=0.015). Death from a non-infectious pulmonary cause was more frequent among patients with a TERT rare variant. The majority of variants were missense substitutions and classified as variants of unknown significance (VUS). Therefore, we cloned all rare missense variants and quantified their impact on telomere elongation in a cell-based assay. We found that 90 percent of TERT rare variants had severe or intermediate impairment in their capacity to elongate telomeres. Using a homology model of human TERT bound to the shelterin protein TPP1, we inferred that TERT rare variants disrupt domain-specific functions, including catalysis, protein-RNA interactions, and recruitment to telomeres. Our results indicate that the contribution of TERT rare variants to MDS pathogenesis and NRM risk is underrecognized. Routine screening for TERT rare variants in MDS patients regardless of age or clinical suspicion may identify clinically inapparent telomere biology disorders and improve transplant outcomes through risk-adapted approaches.

The identification of Rac1 palmitoylation reveals how Rac1 changes subcellular localization to modulate its signalling function.