Radiofrequency Ablation of Parathyroid Glands to Treat a Patient With Hypercalcemia Caused by a Novel Inactivating Mutation in CaSR

ObjectiveWe identified a novel inactivating mutation in the calcium-sensing receptor (CaSR) gene in a patient with refractory hypocalciuric hypercalcemia and analyzed its function. The effectiveness of radiofrequency ablation of the parathyroid glands to treat hypercalcemia caused by this mutation was explored.MethodsClinical data of patients before and after radiofrequency ablation were retrospectively analyzed. The CaSR mutation (D99N) found in the patient was studied in cell lines. HEK-293 cells were transfected with plasmids containing wild-type (WT) or mutant CaSR genes (D99N and W718X). Expression levels of the respective CaSR proteins were measured, and their functions were assessed by examining the effect of NPS R-568 (a CaSR agonist) on intracellular Ca2+ oscillations and that of exogenous parathyroid hormone (PTH) on intracellular cyclic adenosine monophosphate (cAMP) levels.ResultsThe effectiveness of pharmacological treatment was poor, whereas radiofrequency ablation of the parathyroid glands resulted in controlled blood calcium and PTH levels in the patient. In cell lines, upon NPS R-568 administration, the amplitude of intracellular Ca2+ oscillations in the D99N group was lower than that in the WT group and higher than that in the W718X group. Upon administration of PTH, intracellular cAMP levels in the D99N group were higher than those in the WT group and lower than those in the W718X group.ConclusionThe homozygous mutation D99N reduced CaSR activity and caused more severe hypocalciuric hypercalcemia. For patients with this type of hypercalcemia and poor response to pharmacological treatments, radiofrequency ablation of the parathyroid glands may be a suitable treatment option.

CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

Magnesium is essential for cellular life, but how it is homeostatically controlled still remains poorly understood. Here, we report that members of CNNM family, which have been controversially implicated in both cellular Mg2+ influx and efflux, selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells. Coexpression of CNNMs with the channel markedly increased uptake of divalent cations, which is prevented by an inactivating mutation to the channel’s pore. Knockout (KO) of Trpm7 in cells or application of the TRPM7 channel inhibitor NS8593 also interfered with CNNM-stimulated divalent cation uptake. Conversely, KO of CNNM3 and CNNM4 in HEK-293 cells significantly reduced TRPM7-mediated divalent cation entry, without affecting TRPM7 protein expression or its cell surface levels. Furthermore, we found that cellular overexpression of phosphatases of regenerating liver (PRLs), known CNNMs binding partners, stimulated TRPM7-dependent divalent cation entry and that CNNMs were required for this activity. Whole-cell electrophysiological recordings demonstrated that deletion of CNNM3 and CNNM4 from HEK-293 cells interfered with heterologously expressed and native TRPM7 channel function. We conclude that CNNMs employ the TRPM7 channel to mediate divalent cation influx and that CNNMs also possess separate TRPM7-independent Mg2+ efflux activities that contribute to CNNMs’ control of cellular Mg2+ homeostasis.

Molecular Dissection of the Primase and Polymerase Activities of Deep-Sea Phage NrS-1 Primase-Polymerase

PrimPols are a class of primases that belong to the archaeo-eukaryotic primase (AEP) superfamily but have both primase and DNA polymerase activities. Replicative polymerase from NrS-1 phage (NrSPol) is a representative of the PrimPols. In this study, we identified key residues for the catalytic activity of NrSPol and found that a loop in NrSPol functionally replaces the zinc finger motif that is commonly found in other AEP family proteins. A helix bundle domain (HBD), conserved in the AEP superfamily, was recently reported to bind to the primase recognition site and to be crucial for initiation of primer synthesis. We found that NrSPol can recognize different primase recognition sites, and that the initiation site for primer synthesis is not stringent, suggesting that the HBD conformation is flexible. More importantly, we found that although the HBD-inactivating mutation impairs the primase activity of NrSPol, it significantly enhances the DNA polymerase activity, indicating that the HBD hinders the DNA polymerase activity. The conflict between the primase activity and the DNA polymerase activity in a single protein with the same catalytic domain may be one reason for why DNA polymerases are generally unable to synthesize DNA de novo.

The VINE complex is a VPS9–domain GEF–containing SNX–BAR coat involved in endosomal sorting

Membrane trafficking pathways perform important roles in establishing and maintaining the endolysosomal network. Retrograde protein sorting from the endosome is promoted by conserved SNX–BAR–containing coat complexes including retromer which enrich cargo at tubular microdomains and generate transport carriers. In metazoans, retromer cooperates with VARP, a conserved VPS9–domain GEF, to direct an endosomal recycling pathway. The function of the yeast VARP homolog Vrl1 has been overlooked due an inactivating mutation in commonly studied strains. Here, we demonstrate that Vrl1 has features of a SNX–BAR coat protein and forms an obligate complex with Vin1, the paralog of the retromer SNX–BAR protein Vps5. Unique features in the Vin1 N–terminus allow Vrl1 to distinguish it from Vps5, thereby forming what we have named the VINE complex. VINE occupies endosomal tubules and promotes the delivery of a conserved mannose 6–phosphate receptor–like protein to the vacuolar membrane. In addition to sorting functions, membrane recruitment by Vin1 is essential for Vrl1 GEF activity, suggesting that VINE is a multifunctional coat complex that regulates trafficking and signaling events at the endosome.

Michaelis-like complex of SARS-CoV-2 main protease visualized by room-temperature X-ray crystallography

SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of Mpro, a cysteine protease with a catalytic site comprising the noncanonical Cys145–His41 dyad, can help in guiding drug design. Here, a 2.0 Å resolution room-temperature X-ray crystal structure is reported of a Michaelis-like complex of Mpro harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH2 corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3′ by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free Mpro demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for in silico modeling of the SARS-CoV-2 Mpro catalytic mechanism.

Familial Hypocalciuric Hypercalcemia: The Challenge of Diagnosis

Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant genetic disorder classically characterized by lifelong mild-to-moderate asymptomatic hypercalcemia with inappropriately normal to elevated serum parathyroid hormone (PTH) concentrations and hypocalciuria, best expressed by a urine calcium-to-creatinine clearance ratio (CCCR)<0.01[1,2]. FHH prevalence is estimated between 1:10 000 to 1:100 000[3,4]. In 60% of cases, FHH is due to CASR inactivating mutation[5]. More rarely FHH is due to AP2S1 or GNA11 inactivating mutation, both genes encoding for proteins involved downstream of CASR activation[6]. These molecular alterations are found in all parathyroid cells, explaining disease persistence following partial parathyroidectomy and the ineffective surgical management of these patients. FHH phenotypes could however overlap with primary hyperparathyroidism (PHPT). Indeed, even if patients with FHH are currently asymptomatic, some of them present chondrocalcinosis, kidney stones or bone fracture and very high level of PTH or calcemia[7]. Nonetheless, the distinction has to be adressed since the therapeutic approach significantly differs between these two conditions. Surgery is usually recommended for PHPT[8] while follow-up is preferred in the latter case[9,10]. We report and discuss 7 cases, 6 out 7 being operated for a presumed PHPT.

Case Report: Two Monochorionic Twins With a Critically Different Course of Progressive Osseus Heteroplasia

Progressive osseous heteroplasia (POH; OMIM 166350) is a rare autosomal-dominant genetic disorder in which extra-skeletal bone forms within skin and muscle tissue. POH is one of the clinical manifestations of an inactivating mutation in the GNAS gene. GNAS gene alterations are difficult matter to address, as GNAS alleles show genetic imprinting and produce several transcript products, and the same mutation may lead to strikingly different phenotypes. Also, most of the publications concerning POH patients are either clinical depictions of a case (or a case series), descriptions of their genetic background, or a tentative correlation of both clinical and molecular findings. Treatment for POH is rarely addressed, and POH still lacks therapeutic options. We describe a unique case of POH in two monochorionic twins, who presented an almost asymptomatic vs. the severe clinical course, despite sharing the same mutation and genetic background. We also report the results of the therapeutic interventions currently available for heterotopic ossification in the patient with the severe course. This article not only critically supports the assumption that the POH course is strongly influenced by factors beyond genetic background but also remarks the lack of options for patients suffering an orphan disease, even after testing drugs with promising in vitro results.

Hypercalcemia With Non Suppressed Parathyroid Hormone in a Young Female- A Rare Case of Calcium Sensing Receptor Gene Related Familial Hypocalciuric Hypercalcemia-1

Background: Familial Hypocalciuric Hypercalcemia (FHH) is a rare disorder, associated with hypercalcemia and hypocalciuria and inherited in an autosomal dominant manner. Its true prevalence is unknown, and is estimated to be around 1 in 78,000 as compared to primary hyperparathyroidism (PHPT) which is around 1 in 1000. There are 3 mutations known to cause FHH. FHH-1 is caused by inactivating mutation in the calcium sensing receptor (CaSR) gene. Mutations associated with FHH-2 and FHH-3 are GNA11 and AP2S1 respectively. Case Presentation: 38-year-old female presented to endocrinology, for evaluation of hypercalcemia (10.8 mg/dL, with normal albumin). She was not on any calcium supplements or any other medications which can cause hypercalcemia. She did not have any prior fracture or nephrolithiasis or renal insufficiency. Her father reported to have hypercalcemia; no further evaluations of her father were available. Examination revealed an obese female with no skeletal or dental or other physical abnormalities. Laboratory evaluations: PTH= 37 pg/ml (15–65), 25 OH vitamin=D-30 pg/ml, Mg=2 mg/dL (1.5–2.6), phosphorus=3.1 mg/dl (2.5–4.8), 24 hour urine calcium=0.151 g/24 hours-with 24 hour urine calcium clearance of 0.008. Therefore, evaluations were highly suggestive of FHH. Subsequently, she had genetic evaluation which confirmed heterozygous CaSR mutation, confirming FHH-1. Her mother had genetic testing and was not found to have the mutation. So, it was concluded that the patient likely inherited the gene from her father, who also had hypercalcemia. She is being monitored clinically and with serial laboratory evaluations to monitor her calcium levels. Discussion: CaSR is expressed in parathyroid glands and kidneys which plays a key role in calcium regulation. CaSR inactivating mutation (seen in FHH-1) leads to hypocalciuria and hypercalcemia. 24 hour urine calcium clearance (urine Ca x serum Cr/ serum Ca x urine Cr) of &lt;0.01 is highly suggestive of FHH. Furthermore, higher concentration of calcium is required to suppress PTH release leading to high or nonsuppressed PTH. This finding can mislead towards the diagnosis of PHPT and unnecessary parathyroid surgery, if the diagnosis of FHH is missed. FHH is usually a benign disorder; subtotal parathyroidectomy does not cure the disease. FHH, rarely can cause atypical complications such as pancreatitis and total parathyroidectomy may be indicated to prevent further episodes of pancreatitis. Conclusions: This is a rare case presentation of hypercalcemia due to CaSR inactivating mutation related FHH. FHH and PHPT are competing diagnoses, when a patient presents with hypercalcemia and has nonsuppressed PTH. FHH is rare, however needs to be suspected in a young patient with family history of hypercalcemia, to avoid misdiagnosis of PHPT and unnecessary surgical intervention.

KEAP1 Is Required for Artesunate Anticancer Activity in Non-Small-Cell Lung Cancer

Artesunate is the most common treatment for malaria throughout the world. Artesunate has anticancer activity likely through the induction of reactive oxygen species, the same mechanism of action utilized in Plasmodium falciparum infections. Components of the kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway, which regulates cellular response to oxidative stress, are mutated in approximately 30% of non-small-cell lung cancers (NSCLC); therefore, we tested the hypothesis that KEAP1 is required for artesunate sensitivity in NSCLC. Dose response assays identified A549 cells, which have a G333C-inactivating mutation in KEAP1, as resistant to artesunate, with an IC50 of 23.6 µM, while H1299 and H1563 cells were sensitive to artesunate, with a 10-fold lower IC50. Knockdown of KEAP1 through siRNA caused increased resistance to artesunate in H1299 cells. Alternatively, the pharmacological inhibition of NRF2, which is activated downstream of KEAP1 loss, by ML385 partially restored sensitivity of A549 cells to artesunate, and the combination of artesunate and ML385 was synergistic in both A549 and H1299 cells. These findings demonstrate that KEAP1 is required for the anticancer activity of artesunate and support the further development of NRF2 inhibitors to target patients with mutations in the KEAP1/NRF2 pathway.