Inhibitors of the tyrosine kinases FMS-like tyrosine kinase-3 and WEE1 induce apoptosis and DNA damage synergistically in acute myeloid leukemia cells.

Hyperactive FMS-like receptor tyrosine kinase-3 mutants with internal tandem duplications (FLT3-ITD) are frequent driver mutations of aggressive acute myeloid leukemia (AML). Inhibitors of FLT3 produce promising results in rationally designed cotreatment schemes. Since FLT3-ITD modulates DNA replication and DNA repair, valid anti-leukemia strategies could rely on a combined inhibition of FLT3-ITD and regulators of cell cycle progression and DNA integrity. These include the WEE1 kinase which controls cell cycle progression, nucleotide synthesis, and DNA replication origin firing. We investigated how pharmacological inhibition of FLT3 and WEE1 affected the survival and genomic integrity of AML cell lines and primary AML cells. We reveal that promising clinical grade and preclinical inhibitors of FLT3 and WEE1 synergistically trigger apoptosis in leukemic cells that express FLT3-ITD. An accumulation of single and double strand DNA damage precedes this process. Mass spectrometry-based proteomic analyses show that FLT3-ITD and WEE1 sustain the expression of the ribonucleotide reductase subunit RRM2, which provides dNTPs for DNA replication. Unlike their strong pro-apoptotic effects on leukemia cells with FLT3-ITD, inhibitors of FLT3 and WEE1 do not damage healthy human blood cells and murine hematopoietic stem cells. Thus, pharmacological inhibition of FLT3-ITD and WEE1 might become an improved, rationally designed therapeutic option.

Replenishing IRAK-M expression in retinal pigment epithelium attenuates outer retinal degeneration.

Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor-associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants of IRAK3, which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD. Irak3-knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)-expressing human IRAK3 rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in Irak3-knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.

The protein phosphatase-2A subunit PR130 is involved in the formation of cytotoxic protein aggregates in pancreatic ductal adenocarcinoma cells.

As a major source of cellular serine and threonine phosphatase activity, protein phosphatase-2A (PP2A) modulates signaling pathways in health and disease. PP2A complexes consist of catalytic, scaffolding, and B-type subunits. Seventeen PP2A B-type subunits direct PP2A complexes to selected substrates. It is ill-defined how PP2A B-type subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis. We analyzed the responses of murine and human mesenchymal and epithelial PDAC cells to the specific PP2A inhibitor phendione. We assessed protein levels by immunoblot and proteomics and cell fate by flow cytometry, confocal microscopy, and genetic manipulation. We show that murine mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 than epithelial PDAC cells. This overexpression of PR130 is associated with a dependency of such metastasis-prone cells on the catalytic activity of PP2A. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in murine mesenchymal and human PDAC cells. These processes occur independently of the frequently mutated tumor suppressor p53. Proteomic analyses reveal that phendione upregulates the chaperone HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis and phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes murine and human PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data suggest that the PP2A-PR130 complex dephosphorylates and thereby prevents the aggregation of proteins in tumor cells.

Pharmacological degradation of ATR induces antiproliferative DNA replication stress in leukemic cells.

Mammalian cells replicate ~ 3 × 109 base pairs per cell cycle. One of the key molecules that slows down the cell cycle and prevents excessive DNA damage upon DNA replication stress is the checkpoint kinase ataxia-telangiectasia-and-RAD3-related (ATR). Proteolysis-targeting-chimeras (PROTACs) are an innovative pharmacological invention to molecularly dissect, biologically understand, and therapeutically assess catalytic and non-catalytic functions of enzymes. This work defines the first-in-class ATR PROTAC, Abd110/Ramotac-1. It is derived from the ATR inhibitor VE-821 and recruits the E3 ubiquitin-ligase component cereblon to ATR. Abd110 eliminates ATR rapidly in human leukemic cells. This mechanism provokes DNA replication catastrophe and augments anti-leukemic effects of the clinically used ribonucleotide reductase-2 inhibitor hydroxyurea. Moreover, Abd110 is more effective than VE-821 against human primary leukemic cells but spares normal primary immune cells. CRISPR-Cas9 screens show that ATR is a dependency factor in 116 myeloid and lymphoid leukemia cells. Treatment of wild-type but not of cereblon knockout cells with Abd110 stalls their proliferation which verifies that ATR elimination is the primary mechanism of Abd110. Altogether, our findings demonstrate specific anti-leukemic effects of an ATR PROTAC.

Design, synthesis, and biological characterization of proteolysis targeting chimera (PROTACs) for the ataxia telangiectasia and RAD3-related (ATR) kinase.

The Ataxia telangiectasia and RAD3-related (ATR) kinase is a key regulator of DNA replication stress responses and DNA-damage checkpoints. Several potent and selective ATR inhibitors are reported and four of them are currently in clinical trials in combination with radio- or chemotherapy. Based on the idea of degrading target proteins rather than inhibiting them, we designed, synthesized and biologically characterized a library of ATR-targeted proteolysis targeting chimera (PROTACs). Among the synthesized compounds, the lenalidomide-based PROTAC 42i was the most promising. In pancreatic and cervix cancer cells cancer cells, it reduced ATR to 40 % of the levels in untreated cells. 42i selectively degraded ATR through the proteasome, dependent on the E3 ubiquitin ligase component cereblon, and without affecting the associated kinases ATM and DNA-PKcs. 42i may be a promising candidate for further optimization and biological characterization in various cancer cells.

Autophagy facilitates intracellular survival of pathogenic rickettsiae in macrophages via evasion of autophagosomal maturation and reduction of microbicidal pro-inflammatory IL-1 cytokine responses.

IMPORTANCE: Rickettsia spp. are intracellular bacterial parasites of a wide range of arthropod and vertebrate hosts. Some rickettsiae are responsible for several severe human diseases globally. One interesting feature of these pathogens is their ability to exploit host cytosolic defense responses to their benefits. However, the precise mechanism by which pathogenic Rickettsia spp. elude host defense responses remains unclear. Here, we observed that pathogenic Rickettsia typhi and Rickettsia rickettsii (Sheila Smith [SS]), but not non-pathogenic Rickettsia montanensis, become ubiquitinated and induce autophagy upon entry into macrophages. Moreover, unlike R. montanensis, R. typhi and R. rickettsii (SS) colocalized with LC3B but not with Lamp2 upon host cell entry. Finally, we observed that both R. typhi and R. rickettsii (SS), but not R. montanensis, reduce pro-inflammatory interleukin-1 (IL-1) responses, likely via an autophagy-mediated mechanism. In summary, we identified a previously unappreciated pathway by which both pathogenic R. typhi and R. rickettsii (SS) become ubiquitinated, induce autophagy, avoid autolysosomal destruction, and reduce microbicidal IL-1 cytokine responses to establish an intracytosolic niche in macrophages.

Medicinal chemistry advances in targeting class I histone deacetylases.

Histone deacetylases (HDACs) are a class of zinc (Zn)-dependent metalloenzymes that are responsible for epigenetic modifications. HDACs are largely associated with histone proteins that regulate gene expression at the DNA level. This tight regulation is controlled by acetylation [via histone acetyl transferases (HATs)] and deacetylation (via HDACs) of histone and non-histone proteins that alter the coiling state of DNA, thus impacting gene expression as a downstream effect. For the last two decades, HDACs have been studied extensively and indicated in a range of diseases where HDAC dysregulation has been strongly correlated with disease emergence and progression-most prominently, cancer, neurodegenerative diseases, HIV, and inflammatory diseases. The involvement of HDACs as regulators in these biochemical pathways established them as an attractive therapeutic target. This review summarizes the drug development efforts exerted to create HDAC inhibitors (HDACis), specifically class I HDACs, with a focus on the medicinal chemistry, structural design, and pharmacology aspects of these inhibitors.

NOXA Accentuates Apoptosis Induction by a Novel Histone Deacetylase Inhibitor.

Epigenetic modifiers of the histone deacetylase (HDAC) family are often dysregulated in cancer cells. Experiments with small molecule HDAC inhibitors (HDACi) have proven that HDACs are a vulnerability of transformed cells. We evaluated a novel hydroxamic acid-based HDACi (KH16; termed yanostat) in human pancreatic ductal adenocarcinoma (PDAC) cells, short- and long-term cultured colorectal cancer (CRC) cells, and retinal pigment epithelial cells. We show that KH16 induces cell cycle arrest and apoptosis, both time and dose dependently in PDAC and CRC cells. This is associated with altered expression of BCL2 family members controlling intrinsic apoptosis. Recent data illustrate that PDAC cells frequently have an altered expression of the pro-apoptotic BH3-only protein NOXA and that HDACi induce an accumulation of NOXA. Using PDAC cells with a deletion of NOXA by CRISPR-Cas9, we found that a lack of NOXA delayed apoptosis induction by KH16. These results suggest that KH16 is a new chemotype of hydroxamic acid HDACi with superior activity against solid tumor-derived cells. Thus, KH16 is a scaffold for future research on compounds with nanomolar activity against HDACs.

Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells.

INTRODUCTION: Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers. OBJECTIVES: We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1,-2,-3,-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells. METHODS: We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells. RESULTS: Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (vorinostat). Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC50 = 6 nM) and the most potent inducer of apoptosis (IC50 = 110 nM; p < 0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in 115 human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces caspase-dependent processing of the autophagy proteins ULK1 and p62. CONCLUSION: These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention.

Skin cancer, sunscreens, and prevention: discussions to have with patients.

With the incidence of skin cancer increasing in the United States, the role of dental providers in noting suspicious skin lesions as well as advising patients in proper sun-protective strategies is more important than ever. However, the public conversation about proper sun protection is often laden with misunderstandings and pervasive myths. This article reviews the most common types of skin cancer, elucidates the differences among the types of UV radiation, and offers updated recommendations for proper sunscreen use along with adjunctive sun-protective strategies.

Thermoregulatory trade-offs underlie the effects of warming summer temperatures on deer mice.

Climate warming could challenge the ability of endotherms to thermoregulate and maintain normal body temperature (Tb), but the effects of warming summer temperatures on activity and thermoregulatory physiology in many small mammals remain poorly understood. We examined this issue in deer mice (Peromyscus maniculatus), an active nocturnal species. Mice were exposed in the lab to simulated seasonal warming, in which an environmentally realistic diel cycle of ambient temperature (Ta) was gradually warmed from spring conditions to summer conditions (controls were maintained in spring conditions). Activity (voluntary wheel running) and Tb (implanted bio-loggers) were measured throughout, and indices of thermoregulatory physiology (thermoneutral zone, thermogenic capacity) were assessed after exposure. In control mice, activity was almost entirely restricted to the night-time, and Tb fluctuated ∼1.7°C between daytime lows and night-time highs. Activity, body mass and food consumption were reduced and water consumption was increased in later stages of summer warming. This was accompanied by strong Tb dysregulation that culminated in a complete reversal of the diel pattern of Tb variation, with Tb reaching extreme highs (∼40°C) during daytime heat but extreme lows (∼34°C) at cooler night-time temperatures. Summer warming was also associated with reduced ability to generate body heat, as reflected by decreased thermogenic capacity and decreased mass and uncoupling protein (UCP1) content of brown adipose tissue. Our findings suggest that thermoregulatory trade-offs associated with daytime heat exposure can affect Tb and activity at cooler night-time temperatures, impacting the ability of nocturnal mammals to perform behaviours important for fitness in the wild.

Pharmacological Modulation of the Crosstalk between Aberrant Janus Kinase Signaling and Epigenetic Modifiers of the Histone Deacetylase Family to Treat Cancer.

Hyperactivated Janus kinase (JAK) signaling is an appreciated drug target in human cancers. Numerous mutant JAK molecules as well as inherent and acquired drug resistance mechanisms limit the efficacy of JAK inhibitors (JAKi). There is accumulating evidence that epigenetic mechanisms control JAK-dependent signaling cascades. Like JAKs, epigenetic modifiers of the histone deacetylase (HDAC) family regulate the growth and development of cells and are often dysregulated in cancer cells. The notion that inhibitors of histone deacetylases (HDACi) abrogate oncogenic JAK-dependent signaling cascades illustrates an intricate crosstalk between JAKs and HDACs. Here, we summarize how structurally divergent, broad-acting as well as isoenzyme-specific HDACi, hybrid fusion pharmacophores containing JAKi and HDACi, and proteolysis targeting chimeras for JAKs inactivate the four JAK proteins JAK1, JAK2, JAK3, and tyrosine kinase-2. These agents suppress aberrant JAK activity through specific transcription-dependent processes and mechanisms that alter the phosphorylation and stability of JAKs. Pharmacological inhibition of HDACs abrogates allosteric activation of JAKs, overcomes limitations of ATP-competitive type 1 and type 2 JAKi, and interacts favorably with JAKi. Since such findings were collected in cultured cells, experimental animals, and cancer patients, we condense preclinical and translational relevance. We also discuss how future research on acetylation-dependent mechanisms that regulate JAKs might allow the rational design of improved treatments for cancer patients. SIGNIFICANCE STATEMENT: Reversible lysine-ɛ-N acetylation and deacetylation cycles control phosphorylation-dependent Janus kinase-signal transducer and activator of transcription signaling. The intricate crosstalk between these fundamental molecular mechanisms provides opportunities for pharmacological intervention strategies with modern small molecule inhibitors. This could help patients suffering from cancer.

The epigenetic modifier HDAC2 and the checkpoint kinase ATM determine the responses of microsatellite instable colorectal cancer cells to 5-fluorouracil.

The epigenetic modifier histone deacetylase-2 (HDAC2) is frequently dysregulated in colon cancer cells. Microsatellite instability (MSI), an unfaithful replication of DNA at nucleotide repeats, occurs in about 15% of human colon tumors. MSI promotes a genetic frameshift and consequently a loss of HDAC2 in up to 43% of these tumors. We show that long-term and short-term cultures of colorectal cancers with MSI contain subpopulations of cells lacking HDAC2. These can be isolated as single cell-derived, proliferating populations. Xenografted patient-derived colon cancer tissues with MSI also show variable patterns of HDAC2 expression in mice. HDAC2-positive and HDAC2-negative RKO cells respond similarly to pharmacological inhibitors of the class I HDACs HDAC1/HDAC2/HDAC3. In contrast to this similarity, HDAC2-negative and HDAC2-positive RKO cells undergo differential cell cycle arrest and apoptosis induction in response to the frequently used chemotherapeutic 5-fluorouracil, which becomes incorporated into and damages RNA and DNA. 5-fluorouracil causes an enrichment of HDAC2-negative RKO cells in vitro and in a subset of primary colorectal tumors in mice. 5-fluorouracil induces the phosphorylation of KAP1, a target of the checkpoint kinase ataxia-telangiectasia mutated (ATM), stronger in HDAC2-negative cells than in their HDAC2-positive counterparts. Pharmacological inhibition of ATM sensitizes RKO cells to cytotoxic effects of 5-fluorouracil. These findings demonstrate that HDAC2 and ATM modulate the responses of colorectal cancer cells towards 5-FU.

99mTc­labeled single-domain antibody for SPECT/CT assessment of HER2 expression in diverse cancer types.

The expression status of human epidermal growth factor receptor 2 (HER2) in cancer predicts response to HER2-targeted therapy. Therefore, its accurate determination is of utmost importance. In recent years, there has been an increase in research on noninvasive techniques for molecular imaging, as this method offers the advantages of a more accurate determination of HER2 status without the need for multiple biopsies. The technetium-labeled single-domain antibody RAD201, previously known as 99mTc-NM-02, has been shown to be safe for use in breast cancer imaging with reasonable radiation doses, favorable biodistribution, and imaging characteristics. METHODS: A total of six HER2-positive, heavily pretreated patients with different cancer types aged between 42 and 69 years (5 women and 1 man; the median age of 55.5) have been examined. In six of seven scans, the patients were administered 500 ml of Gelofusine® solution (40 mg/ml) for radiation protection before the tracer injection (434 ± 42 MBq). Planar scans were acquired with the patient supine at 10 min, 60 min, 160 min, 20 h, and 24 h after injection. A CT scan was acquired at 95 min, followed by local tomographic SPECT imaging. RESULTS: One patient was scanned twice with RAD201, 3 months apart, resulting in a total of seven scans for six patients. Here, we show that the use of RAD201 in our patient group shows the same favorable biodistribution as in a previous study with RAD201 (NCT04040686) and that the radiation dose to the critical organ kidney can be reduced by the application of the plasma expander Gelofusine® by almost 50%. CONCLUSION: RAD201 appears safe for use in humans and is a promising noninvasive tool for discriminating HER2 status in metastatic (breast) cancer, regardless of ongoing HER2-targeted antibody treatment.

Monitoring Changes in Intracellular Reactive Oxygen Species Levels in Response to Histone Deacetylase Inhibitors.

Reactive oxygen species (ROS) are induced by several chemotherapeutics. In this protocol, we describe a flow cytometry-based method for the analysis of the intracellular levels of ROS in vital leukemic cells in response to the histone deacetylase inhibitor vorinostat. This measurement of ROS using the cell-permeable dye CM-H2DCFDA indicates intracellular oxidative stress.

The synchrotron radiation source PETRA III and its future ultra-low-emittance upgrade PETRA IV.

PETRA III at DESY is one of the brightest synchrotron radiation sources worldwide. It serves a broad international multidisciplinary user community from academia to industry at currently 25 specialised beamlines. With a storage-ring energy of 6 GeV, it provides mainly hard to high-energy X-rays for versatile experiments in a very broad range of scientific fields. It is ideally suited for an upgrade to the ultra-low emittance source PETRA IV, owing to its large circumference of 2304 m. With a targeted storage ring emittance of 20 × 5 pm 2 rad 2 , PETRA IV will reach spectral brightnesses two to three orders of magnitude higher than today. The unique beam parameters will make PETRA IV the ultimate in situ 3D microscope for biological, chemical, and physical processes helping to address key questions in health, energy, mobility, information technology, and earth and environment.

AP1/Fra1 confers resistance to MAPK cascade inhibition in pancreatic cancer.

Targeting KRAS downstream signaling remains an important therapeutic approach in pancreatic cancer. We used primary pancreatic ductal epithelial cells and mouse models allowing the conditional expression of oncogenic KrasG12D, to investigate KRAS signaling integrators. We observed that the AP1 family member FRA1 is tightly linked to the KRAS signal and expressed in pre-malignant lesions and the basal-like subtype of pancreatic cancer. However, genetic-loss-of-function experiments revealed that FRA1 is dispensable for KrasG12D-induced pancreatic cancer development in mice. Using FRA1 gain- and loss-of-function models in an unbiased drug screen, we observed that FRA1 is a modulator of the responsiveness of pancreatic cancer to inhibitors of the RAF-MEK-ERK cascade. Mechanistically, context-dependent FRA1-associated adaptive rewiring of oncogenic ERK signaling was observed and correlated with sensitivity to inhibitors of canonical KRAS signaling. Furthermore, pharmacological-induced degradation of FRA1 synergizes with MEK inhibitors. Our studies establish FRA1 as a part of the molecular machinery controlling sensitivity to MAPK cascade inhibition allowing the development of mechanism-based therapies.

Characteristics of the GlnH and GlnX Signal Transduction Proteins Controlling PknG-Mediated Phosphorylation of OdhI and 2-Oxoglutarate Dehydrogenase Activity in Corynebacterium glutamicum.

In Corynebacterium glutamicum the protein kinase PknG phosphorylates OdhI and thereby abolishes the inhibition of 2-oxoglutarate dehydrogenase activity by unphosphorylated OdhI. Our previous studies suggested that PknG activity is controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX, because ΔglnH and ΔglnX mutants showed a growth defect on glutamine similar to that of a ΔpknG mutant. We have now confirmed the involvement of GlnH and GlnX in the control of OdhI phosphorylation by analyzing the OdhI phosphorylation status and glutamate secretion in ΔglnH and ΔglnX mutants and by characterizing ΔglnX suppressor mutants. We provide evidence for GlnH being a lipoprotein and show by isothermal titration calorimetry that it binds l-aspartate and l-glutamate with moderate to low affinity, but not l-glutamine, l-asparagine, or 2-oxoglutarate. Based on a structural comparison with GlnH of Mycobacterium tuberculosis, two residues critical for the binding affinity were identified and verified. The predicted GlnX topology with four transmembrane segments and two periplasmic domains was confirmed by PhoA and LacZ fusions. A structural model of GlnX suggested that, with the exception of a poorly ordered N-terminal region, the entire protein is composed of α-helices and small loops or linkers, and it revealed similarities to other bacterial transmembrane receptors. Our results suggest that the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade serves to adapt the flux of 2-oxoglutarate between ammonium assimilation via glutamate dehydrogenase and energy generation via the tricarboxylic acid (TCA) cycle to the availability of the amino group donors l-glutamate and l-aspartate in the environment. IMPORTANCE Actinobacteria comprise a large number of species playing important roles in biotechnology and medicine, such as Corynebacterium glutamicum, the major industrial amino acid producer, and Mycobacterium tuberculosis, the pathogen causing tuberculosis. Many actinobacteria use a signal transduction process in which the phosphorylation status of OdhI (corynebacteria) or GarA (mycobacteria) regulates the carbon flux at the 2-oxoglutarate node. Inhibition of 2-oxoglutarate dehydrogenase by unphosphorylated OdhI shifts the flux of 2-oxoglutarate from the TCA cycle toward glutamate formation and, thus, ammonium assimilation. Phosphorylation of OdhI/GarA is catalyzed by the protein kinase PknG, whose activity was proposed to be controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX. In this study, we combined genetic, biochemical, and structural modeling approaches to characterize GlnH and GlnX of C. glutamicum and confirm their roles in the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade. These findings are relevant also to other Actinobacteria employing a similar control process.