Understanding the Logistics for the Distribution of Heme in Cells.

Heme is essential for the survival of virtually all living systems-from bacteria, fungi, and yeast, through plants to animals. No eukaryote has been identified that can survive without heme. There are thousands of different proteins that require heme in order to function properly, and these are responsible for processes such as oxygen transport, electron transfer, oxidative stress response, respiration, and catalysis. Further to this, in the past few years, heme has been shown to have an important regulatory role in cells, in processes such as transcription, regulation of the circadian clock, and the gating of ion channels. To act in a regulatory capacity, heme needs to move from its place of synthesis (in mitochondria) to other locations in cells. But while there is detailed information on how the heme lifecycle begins (heme synthesis), and how it ends (heme degradation), what happens in between is largely a mystery. Here we summarize recent information on the quantification of heme in cells, and we present a discussion of a mechanistic framework that could meet the logistical challenge of heme distribution.

Unravelling the mechanisms controlling heme supply and demand.

In addition to heme's role as the prosthetic group buried inside many different proteins that are ubiquitous in biology, there is new evidence that heme has substantive roles in cellular signaling and regulation. This means that heme must be available in locations distant from its place of synthesis (mitochondria) in response to transient cellular demands. A longstanding question has been to establish the mechanisms that control the supply and demand for cellular heme. By fusing a monomeric heme-binding peroxidase (ascorbate peroxidase, mAPX) to a monomeric form of green-fluorescent protein (mEGFP), we have developed a heme sensor (mAPXmEGFP) that can respond to heme availability. By means of fluorescence lifetime imaging, this heme sensor can be used to quantify heme concentrations; values of the mean fluorescence lifetime (τMean) for mAPX-mEGFP are shown to be responsive to changes in free (unbound) heme concentration in cells. The results demonstrate that concentrations are typically limited to one molecule or less within cellular compartments. These miniscule amounts of free heme are consistent with a system that sequesters the heme and is able to buffer changes in heme availability while retaining the capability to mobilize heme when and where it is needed. We propose that this exchangeable supply of heme can operate using mechanisms for heme transfer that are analogous to classical ligand-exchange mechanisms. This exquisite control, in which heme is made available for transfer one molecule at a time, protects the cell against the toxic effect of excess heme and offers a simple mechanism for heme-dependent regulation in single-molecule steps.

Metformin-associated Encephalopathy in Hemodialysis.

Metformin-associated encephalopathy in maintenance hemodialysis is very rare in literature, till now only three to four cases are published. We report a patient on maintenance hemodialysis from standalone unit presented to us with abnormal neurological signs and symptoms. His medication chart included metformin, which he was taking for quite a long time. Computed tomography brain showed hypointensity in bilateral basal ganglia. Magnetic resonance imaging (MRI) brain showed hyperintensity in T2/fluid-attenuated inversion recovery sequences suggestive of Lentiform fork sign. We stopped metformin, and he was continued on regular hemodialysis. He showed dramatic improvement in neurological manifestations. Two months later, we repeated MRI brain, which showed resolution of basal ganglia changes. We should suspect the possibility of this when a diabetic end-stage renal disease presents with unknown etiology of encephalopathy.

Precise determination of heme binding affinity in proteins.

Accumulating evidence suggests a new role for cellular heme as a signalling molecule, in which interactions with target proteins are more transient than found with traditionally-defined hemoproteins. To study this role, a precise method is needed for determining the heme-binding affinity (or dissociation constant, Kd). Estimates of Kd are commonly made following a spectrophotometric titration of an apo-protein with hemin. An impediment to precise determination is, however, the challenge in discriminating between the Soret absorbance for the product (holo-protein) and that for the titrant (hemin). An altogether different approach has been used in this paper to separate contributions made by these components to absorbance values. The pure component spectra and concentration profiles are estimated by a multivariate curve-resolution (MCR) algorithm. This approach has significant advantages over existing methods. First, a more precise determination of Kd can be made as concentration profiles for all three components (apo-protein/holo-protein/hemin) are determined and can be simultaneously fitted to a theoretical-binding model. Second, an absorption spectrum for the holo-protein is calculated. This is a unique advantage of MCR and attractive for investigating proteins in which the nature of heme binding has not, hitherto, been characterised because the holo-protein spectrum provides information on the interaction.

Capillaroscopic findings in a case of Hajdu-Cheney syndrome.

Hajdu-Cheney syndrome (HCS) is a rare disease which causes osteoporosis, digit shortening, and early tooth loss. In a young HCS female patient, the nailfold capillaroscopy showed reduced capillary height and reduced density in all affected fingers. Capillaroscopy could improve follow-up and therapy assessment in HCS. Hajdu-Cheney syndrome (HCS) is a very rare connective tissue disease characterized by osteoporosis, early dentition loss and a particular phenotype as a result of enhanced NOTCH2 signaling. The pathogenesis of bone resorption and osteoporosis is not fully understood. The altered angiogenesis may play a role in acroosteolysis. We performed capillaroscopy in order to assess the microvascular involvement in a 21-year-old female patient with sporadic HCS. The patient presented with severe parodontopathy, acroosteolysis, and clubbing of four fingers and three toes. Hand radiographs showed periarticular osteoporosis and asymmetric bony involvement with acral resorption and/or transversal lucency bands in several fingers. Early collagen-vascular diseases were ruled out by clinical and ancillary examinations, including immunology and immunoblot for systemic sclerosis. Nailfold capillaroscopy showed reduction of capillary height and density in all affected fingers. Notably, in the fingers with acral resorption, many capillaries were dilated, while in the ones with radiolucency band, capillary dilation was a rare finding. In clinically unaffected fingers, the capillaroscopic findings were normal.To our knowledge, this is the first report of capillaroscopic findings in HCS. The nailfold capillaroscopic aspect reflects the involvement of acral vessels in HCS; thus, capillaroscopy may represent an early diagnostic tool as well as a means of therapeutical assessment. Repeated capillaroscopy in HCS may also add to the understanding of its pathogenesis.