Jörg Heierhorst

Cardiac S100A1 Protein Levels Determine Contractile Performance and Propensity Toward Heart Failure After Myocardial Infarction

Background— Diminished cardiac S100A1 protein levels are characteristic of ischemic and dilated human cardiomyopathy. Because S100A1 has recently been identified as a Ca2+-dependent inotropic factor in the heart, this study sought to explore the pathophysiological relevance of S100A1 levels in development and progression of postischemic heart failure (HF). Methods and Results— S100A1-transgenic (STG) and S100A1-knockout (SKO) mice were subjected to myocardial infarction (MI) by surgical left anterior descending coronary artery ligation, and survival, cardiac function, and remodeling were compared with nontransgenic littermate control (NLC) and wild-type (WT) animals up to 4 weeks. Although MI size was similar in all groups, infarcted S100A1-deficient hearts (SKO-MI) responded with acute contractile decompensation and accelerated transition to HF, rapid onset of cardiac remodeling with augmented apoptosis, and excessive mortality. NLC/WT-MI mice, displaying a progressive decrease in cardiac S100A1 expression, showed a later onset of cardiac remodeling and progression to HF. Infarcted S100A1-overexpressing hearts (STG-MI), however, showed preserved global contractile performance, abrogated apoptosis, and prevention from cardiac hypertrophy and HF with superior survival compared with NLC/WT-MI and SKO-MI. Both Gq-protein–dependent signaling and protein kinase C activation resulted in decreased cardiac S100A1 mRNA and protein levels, whereas Gs-protein–related signaling exerted opposite effects on cardiac S100A1 abundance. Mechanistically, sarcoplasmic reticulum Ca2+ cycling and &bgr;-adrenergic signaling were severely impaired in SKO-MI myocardium but preserved in STG-MI. Conclusions— Our novel proof-of-concept study provides evidence that downregulation of S100A1 protein critically contributes to contractile dysfunction of the diseased heart, which is potentially responsible for driving the progressive downhill clinical course of patients with HF.

Expression of the AMP-activated protein kinase β1 and β2 subunits in skeletal muscle

A heterotrimeric member of the AMP‐activated protein kinase (AMPK) isoenzyme family was purified from rat skeletal muscle by immunoaffinity chromatography, consisting of an α2 catalytic and two non‐catalytic subunits, β2 and γ1. The AMPK β2 cDNA (271 amino acids (aa), molecular weight (MW)=30 307, pI 6.3) was cloned from skeletal muscle and found to share an overall identity of 70% with β1 (270 aa, MW=30 475, pI 6.0). In the liver AMPK β1 subunit, Ser‐182 is constitutively phosphorylated whereas in skeletal muscle β2 isoform, we find that Ser‐182 is only partially phosphorylated. In addition, the autophosphorylation sites Ser‐24, Ser‐25 found in the β1 are replaced by Ala‐Glu in the β2 isoform. β2 contains seven more Ser and one less Thr residues than β1, raising the possibility of differential post‐translational regulation. Immunoblot analysis further revealed that soleus muscle (slow twitch) contains exclusively β1 associated with α2, whereas extensor digitorum longus muscle α2 (EDL, fast twitch) associates with β2 as well as β1. Sequence analysis revealed that glycogen synthase, a known AMPK substrate, co‐immunoprecipitated with the AMPK α2β2γ1 complex.

Impaired Cardiac Contractility Response to Hemodynamic Stress in S100A1-Deficient Mice

ABSTRACT Ca2+ signaling plays a central role in cardiac contractility and adaptation to increased hemodynamic demand. We have generated mice with a targeted deletion of the S100A1 gene coding for the major cardiac isoform of the large multigenic S100 family of EF hand Ca2+-binding proteins. S100A1−/− mice have normal cardiac function under baseline conditions but have significantly reduced contraction rate and relaxation rate responses to β-adrenergic stimulation that are associated with a reduced Ca2+ sensitivity. In S100A1−/− mice, basal left-ventricular contractility deteriorated following 3-week pressure overload by thoracic aorta constriction despite a normal adaptive hypertrophy. Surprisingly, heterozygotes also had an impaired response to acute β-adrenergic stimulation but maintained normal contractility in response to chronic pressure overload that coincided with S100A1 upregulation to wild-type levels. In contrast to other genetic models with impaired cardiac contractility, loss of S100A1 did not lead to cardiac hypertrophy or dilation in aged mice. The data demonstrate that high S100A1 protein levels are essential for the cardiac reserve and adaptation to acute and chronic hemodynamic stress in vivo.

Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast.

Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage‐dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell‐cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain‐mediated and damage‐specific BRCT domain–phospho‐H2A‐dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest.

Cyclin-Dependent Kinase 2 Functions in Normal DNA Repair and Is a Therapeutic Target in BRCA1-Deficient Cancers

Abnormal regulation of progression from G(1) to S phase of the cell cycle by altered activity of cyclin-dependent kinases (CDKs) is a hallmark of cancer. However, inhibition of CDKs, particularly CDK2, has not shown selective activity against most cancer cells because the kinase seems to be redundant in control of cell cycle progression. Here, we show a novel role in the DNA damage response and application of CDK inhibitors in checkpoint-deficient cells. CDK2(-/-) mouse fibroblasts and small interfering RNA--mediated or small-molecule--mediated CDK2 inhibition in MCF7 or U2OS cells lead to delayed damage signaling through Chk1, p53, and Rad51. This coincided with reduced DNA repair using the single-cell comet assay and defects observed in both homologous recombination and nonhomologous end-joining in cell-based assays. Furthermore, tumor cells lacking cancer predisposition genes BRCA1 or ATM are 2- to 4-fold more sensitive to CDK inhibitors. These data suggest that inhibitors of CDK2 can be applied to selectively enhance responses of cancer cells to DNA-damaging agents, such as cytotoxic chemotherapy and radiotherapy. Moreover, inhibitors of CDKs may be useful therapeutics in cancers with defects in DNA repair, such as mutations in the familial breast cancer gene BRCA1.

Giant protein kinases: domain interactions and structural basis of autoregulation.

The myosin‐associated giant protein kinases twitchin and titin are composed predominantly of fibronectin‐ and immunoglobulin‐like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C‐terminal immunoglobulin‐like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin‐dependent kinase I.

Diphosphothreonine-specific interaction between an SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade.

Forkhead-associated (FHA) domains recognize phosphothreonines, and SQ/TQ cluster domains (SCDs) contain concentrated phosphorylation sites for ATM/ATR-like DNA-damage-response kinases. The Rad53-SCD1 has dual functions in regulating the activation of the Rad53-Dun1 checkpoint kinase cascade but with unknown molecular mechanisms. Here we present structural, biochemical, and genetic evidence that Dun1-FHA possesses an unprecedented diphosphothreonine-binding specificity. The Dun1-FHA has >100-fold increased affinity for diphosphorylated relative to monophosphorylated Rad53-SCD1 due to the presence of two separate phosphothreonine-binding pockets. In vivo, any single threonine of Rad53-SCD1 is sufficient for Rad53 activation and RAD53-dependent survival of DNA damage, but two adjacent phosphothreonines in the Rad53-SCD1 and two phosphothreonine-binding sites in the Dun1-FHA are necessary for Dun1 activation and DUN1-dependent transcriptional responses to DNA damage. The results uncover a phospho-counting mechanism that regulates the specificity of SCD, and provide mechanistic insight into a role of multisite phosphorylation in DNA-damage signaling.

FHA domains as phospho-threonine binding modules in cell signaling.

Forkhead‐associated (FHA) domains are present in <200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions. Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged. FHA domains form 11‐stranded beta‐sandwiches that contain some 100‐180 amino acid residues with a high degree of sequence diversity. FHA domains act as phosphorylation‐dependent protein‐protein interaction modules that preferentially bind to phospho‐threonine residues in their targets. Interestingly, point mutations in the human CHK2 gene that lead to single‐residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li‐Fraumeni multi‐cancer syndrome. IUBMB Life, 55: 23‐27, 2003

Molecular Basis for Lysine Specificity in the Yeast Ubiquitin-Conjugating Enzyme Cdc34

ABSTRACT Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination.

ASCIZ regulates lesion‐specific Rad51 focus formation and apoptosis after methylating DNA damage

Nuclear Rad51 focus formation is required for homology‐directed repair of DNA double‐strand breaks (DSBs), but its regulation in response to non‐DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain‐containing protein termed ASCIZ that forms Rad51‐containing foci in response to base‐modifying DNA methylating agents but not in response to DSB‐inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus‐like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ‐depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion‐specific focus scaffold in a Rad51‐dependent pathway that resolves cytotoxic repair intermediates, most likely single‐stranded DNA gaps, resulting from MLH1‐dependent processing of base lesions.